hrp conjugated streptavidin  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Thermo Fisher hrp conjugated streptavidin
    Fig. 4. TGase 2 catalyzes polyamination of HPV18 E7, but not HPV16 E7. ( A ) One hundred micrograms of His-tagged HPV18 E7 and HPV16 E7 were incubated with TGase 2 in the presence of 14 C putrescine. Radioactivity bound to each protein was measured by a liquid scintillation counter. BSA and casein were employed as negative and a positive controls respectively. The figure shows the mean values and SD of three independent experiments. ( B ) HPV18 E7 was incubated with TGase 2 in the presence of 0.1 mM of biotinylated spermine and 0, 0.1, 1 and 10 mM of spermine (lane 1–4). The incorporation of biotinylated spermine was analyzed by dot-blotting using <t>HRP-conjugated</t> <t>streptavidin</t> (SA-HRP). HPV18 E7 was probed with anti-HPV18 E7 antibody. ( C ) The reaction mixtures were subjected to 15% SDS–PAGE and polyaminated HPV E7 was probed with SA-HRP. ( D ) HeLa and Caski cells were treated with 1 mM of biotinylated pentylamine for 1 h in the presence of A23187. Biotinylated pentylamine-incorporated E7s were precipitated by streptavidin-conjugated magnetic beads. Proteins bound to beads were analyzed by western blot using antibodies to HPV18 E7 or HPV16 E7. C33A cells were used as a negative control.
    Hrp Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 183 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars

    Images

    1) Product Images from "Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine"

    Article Title: Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg495

    Fig. 4. TGase 2 catalyzes polyamination of HPV18 E7, but not HPV16 E7. ( A ) One hundred micrograms of His-tagged HPV18 E7 and HPV16 E7 were incubated with TGase 2 in the presence of 14 C putrescine. Radioactivity bound to each protein was measured by a liquid scintillation counter. BSA and casein were employed as negative and a positive controls respectively. The figure shows the mean values and SD of three independent experiments. ( B ) HPV18 E7 was incubated with TGase 2 in the presence of 0.1 mM of biotinylated spermine and 0, 0.1, 1 and 10 mM of spermine (lane 1–4). The incorporation of biotinylated spermine was analyzed by dot-blotting using HRP-conjugated streptavidin (SA-HRP). HPV18 E7 was probed with anti-HPV18 E7 antibody. ( C ) The reaction mixtures were subjected to 15% SDS–PAGE and polyaminated HPV E7 was probed with SA-HRP. ( D ) HeLa and Caski cells were treated with 1 mM of biotinylated pentylamine for 1 h in the presence of A23187. Biotinylated pentylamine-incorporated E7s were precipitated by streptavidin-conjugated magnetic beads. Proteins bound to beads were analyzed by western blot using antibodies to HPV18 E7 or HPV16 E7. C33A cells were used as a negative control.
    Figure Legend Snippet: Fig. 4. TGase 2 catalyzes polyamination of HPV18 E7, but not HPV16 E7. ( A ) One hundred micrograms of His-tagged HPV18 E7 and HPV16 E7 were incubated with TGase 2 in the presence of 14 C putrescine. Radioactivity bound to each protein was measured by a liquid scintillation counter. BSA and casein were employed as negative and a positive controls respectively. The figure shows the mean values and SD of three independent experiments. ( B ) HPV18 E7 was incubated with TGase 2 in the presence of 0.1 mM of biotinylated spermine and 0, 0.1, 1 and 10 mM of spermine (lane 1–4). The incorporation of biotinylated spermine was analyzed by dot-blotting using HRP-conjugated streptavidin (SA-HRP). HPV18 E7 was probed with anti-HPV18 E7 antibody. ( C ) The reaction mixtures were subjected to 15% SDS–PAGE and polyaminated HPV E7 was probed with SA-HRP. ( D ) HeLa and Caski cells were treated with 1 mM of biotinylated pentylamine for 1 h in the presence of A23187. Biotinylated pentylamine-incorporated E7s were precipitated by streptavidin-conjugated magnetic beads. Proteins bound to beads were analyzed by western blot using antibodies to HPV18 E7 or HPV16 E7. C33A cells were used as a negative control.

    Techniques Used: Incubation, Radioactivity, SDS Page, Magnetic Beads, Western Blot, Negative Control

    2) Product Images from "Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses"

    Article Title: Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

    Journal: Retrovirology

    doi: 10.1186/1742-4690-5-2

    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.
    Figure Legend Snippet: Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Techniques Used: Expressing

    3) Product Images from "Characterizing the Role of Cell-Wall ?-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37"

    Article Title: Characterizing the Role of Cell-Wall ?-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021394

    Binding of LL-37 to C. albicans cell-wall proteins. (A) Flow cytometry showing that BA-LL37 bound to C. albicans . Cells were treated with 1 mg/ml proteinase K (lower panels) or were not treated (upper panels) prior to incubation with 10 µg BA-LL37. The fluorescence intensity (FL1-H) of SA-DTAF that was associated with the cells via binding to BA-LL37 was measured to determine the amount of BA-LL37 bound to cells. The results are representative of two independent experiments that gave similar results. (B) Extracts prepared by fractionation of C. albicans cell-wall proteins using β-ME and β-glucanase. The proteins in the extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were probed with BA-LL37 and visualized with HRP–conjugated streptavidin. Arrows indicate the three major cell wall proteins bound by LL-37. The positions and values of molecular mass standards are indicated. Data are representative of three independent experiments that gave similar results.
    Figure Legend Snippet: Binding of LL-37 to C. albicans cell-wall proteins. (A) Flow cytometry showing that BA-LL37 bound to C. albicans . Cells were treated with 1 mg/ml proteinase K (lower panels) or were not treated (upper panels) prior to incubation with 10 µg BA-LL37. The fluorescence intensity (FL1-H) of SA-DTAF that was associated with the cells via binding to BA-LL37 was measured to determine the amount of BA-LL37 bound to cells. The results are representative of two independent experiments that gave similar results. (B) Extracts prepared by fractionation of C. albicans cell-wall proteins using β-ME and β-glucanase. The proteins in the extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were probed with BA-LL37 and visualized with HRP–conjugated streptavidin. Arrows indicate the three major cell wall proteins bound by LL-37. The positions and values of molecular mass standards are indicated. Data are representative of three independent experiments that gave similar results.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Incubation, Fluorescence, Fractionation, SDS Page

    4) Product Images from "COVALENT REGULATION OF ULVWF STRING FORMATION AND ELONGATION ON ENDOTHELIAL CELLS UNDER FLOW CONDITIONS †"

    Article Title: COVALENT REGULATION OF ULVWF STRING FORMATION AND ELONGATION ON ENDOTHELIAL CELLS UNDER FLOW CONDITIONS †

    Journal: Journal of thrombosis and haemostasis : JTH

    doi: 10.1111/j.1538-7836.2008.02991.x

    ULVWF did not contain surface exposed free thiols A. ULVWF and plasma VWF multimers were labeled with MPB and immunoprobed with HRP-streptavidin and a polycloncal VWF antibody. Both forms of VWF were labeled with MPB. B. ULVWF and plasma VWF multimers were incubated with the thiol active sepharose 6B beads and the bead captured VWF (containing free thiols) was released by DTT. VWF in supernatant and eluted from the beads was detected by immunoblotting with the polyclonal VWF antibody. The figure is a representative of 6 and 3 separate experiments for plasma VWF and ULVWF, respectively.
    Figure Legend Snippet: ULVWF did not contain surface exposed free thiols A. ULVWF and plasma VWF multimers were labeled with MPB and immunoprobed with HRP-streptavidin and a polycloncal VWF antibody. Both forms of VWF were labeled with MPB. B. ULVWF and plasma VWF multimers were incubated with the thiol active sepharose 6B beads and the bead captured VWF (containing free thiols) was released by DTT. VWF in supernatant and eluted from the beads was detected by immunoblotting with the polyclonal VWF antibody. The figure is a representative of 6 and 3 separate experiments for plasma VWF and ULVWF, respectively.

    Techniques Used: Labeling, Incubation

    5) Product Images from "Disulfide Bond Reduction of Von Willebrand Factor by ADAMTS-13 †"

    Article Title: Disulfide Bond Reduction of Von Willebrand Factor by ADAMTS-13 †

    Journal: Journal of thrombosis and haemostasis : JTH

    doi: 10.1111/j.1538-7836.2010.04094.x

    Surface-exposed thiols in ADAMTS-13 (A) rADAMTS-13 (35 nM) was incubated with thiol beads before and after exposure to 100-dyn/cm 2 shear stress for 3 min at 37°C, separated by 6% SDS-PAGE, and detected by immunoblotting with an anti-Myc antibody. rADAMTS-13 was detected in DTT eluate (thiol form), but not in supernatant (disulfide bond form). (B) rADAMTS-13 was first treated with NEM or IAA for 10 min at RT and subjected to dialysis in 1L of 1XPBS to remove excess NEM or IAA. The treated and untreated rADAMTS-13 was then labeled with MPB, separated by 6% SDS-PAGE, and probed with HRP-streptavidin. (C) Normal human plasma (1 ml) was incubated with 100 mM of MPB or captured by 5 mg of thiol beads for 15–20 min at RT. MPB labeled plasma proteins were precipitated by streptavidin beads, released by boiling in SDS sample buffer, separated on 6% SDS-PAGE, and immunoblotted with an anti-ADAMTS-13 antibody (left lane). ADAMTS-13 captured with thiol beads was released by 20 mM DTT, separated on 6% SDS-PAGE, and blotted with an ADAMTS-13 antibody (middle lane). Unlabeled ADAMTS-13 was used as a control (right lane). (D) Normal human plasma (1 ml) was incubated with MPB before and after it was pretreated with 10 mM of IAA. ADAMTS-13 from labeled plasma was captured by streptavidin-coupled sepharose beads, separated on 6% SDS-PAGE, and immunoblotted with an ADAMTS-13 antibody. Because MPB failed to label IAA-treated plasma, streptavidin beads did not precipitate ADAMTS-13 from plasma (lane 2). Plasma without IAA treatment was used as control. (E) Normal human plasma (1 ml) was incubated with an ADAMTS-13 antibody coupled to protein G sepharose beads before and after it was treated with 10 mM of IAA. The bead-captured plasma ADAMTS-13 was able to release VWF from thiol beads. The figure represents 3–6 separate experiments.
    Figure Legend Snippet: Surface-exposed thiols in ADAMTS-13 (A) rADAMTS-13 (35 nM) was incubated with thiol beads before and after exposure to 100-dyn/cm 2 shear stress for 3 min at 37°C, separated by 6% SDS-PAGE, and detected by immunoblotting with an anti-Myc antibody. rADAMTS-13 was detected in DTT eluate (thiol form), but not in supernatant (disulfide bond form). (B) rADAMTS-13 was first treated with NEM or IAA for 10 min at RT and subjected to dialysis in 1L of 1XPBS to remove excess NEM or IAA. The treated and untreated rADAMTS-13 was then labeled with MPB, separated by 6% SDS-PAGE, and probed with HRP-streptavidin. (C) Normal human plasma (1 ml) was incubated with 100 mM of MPB or captured by 5 mg of thiol beads for 15–20 min at RT. MPB labeled plasma proteins were precipitated by streptavidin beads, released by boiling in SDS sample buffer, separated on 6% SDS-PAGE, and immunoblotted with an anti-ADAMTS-13 antibody (left lane). ADAMTS-13 captured with thiol beads was released by 20 mM DTT, separated on 6% SDS-PAGE, and blotted with an ADAMTS-13 antibody (middle lane). Unlabeled ADAMTS-13 was used as a control (right lane). (D) Normal human plasma (1 ml) was incubated with MPB before and after it was pretreated with 10 mM of IAA. ADAMTS-13 from labeled plasma was captured by streptavidin-coupled sepharose beads, separated on 6% SDS-PAGE, and immunoblotted with an ADAMTS-13 antibody. Because MPB failed to label IAA-treated plasma, streptavidin beads did not precipitate ADAMTS-13 from plasma (lane 2). Plasma without IAA treatment was used as control. (E) Normal human plasma (1 ml) was incubated with an ADAMTS-13 antibody coupled to protein G sepharose beads before and after it was treated with 10 mM of IAA. The bead-captured plasma ADAMTS-13 was able to release VWF from thiol beads. The figure represents 3–6 separate experiments.

    Techniques Used: Incubation, SDS Page, Labeling

    ADAMTS-13 on shear-induced thiol-disulfide exchange of VWF (A) VWF (20 nM) was exposed to a 100-dyn/cm 2 shear stress in the presence and absence of rADAMTS-13 (7 nM) for 3 min at 37°C and incubated with thiol beads for 15–20 min. VWF captured by beads was released by 20 mM DTT and detected by a polyclonal anti-VWF antibody under reducing conditions. (B) VWF multimers were incubated with MPB before and after shear exposure (and with and without rADAMTS-13), and MPB-labeled VWF was detected by HRP-streptavidin. (C) Thiol beads captured VWF before (detected in DTT elute [“e” fraction]), but not after (detected in supernatant [“s” fraction]) being exposed to fluid shear stress in the presence of 1% BSA. (D) VWF was exposed to 100 dyn/cm 2 shear stress in the presence (lane 1) or absence (lane 2) of 7 nM of rADAMTS-13, separated on 6% SDS-PAGE, and immunoblotted with a polyclonal VWF antibody. As a control, VWF was subjected to a standard static assay of VWF cleavage at a low–ionic-strength in the presence of 1 mM BaCl 2 and 1.5 M urea for 0 (lane 3) and 16 hrs (lane 4) at 37°C. The figure represents 3-5 separate experiments.
    Figure Legend Snippet: ADAMTS-13 on shear-induced thiol-disulfide exchange of VWF (A) VWF (20 nM) was exposed to a 100-dyn/cm 2 shear stress in the presence and absence of rADAMTS-13 (7 nM) for 3 min at 37°C and incubated with thiol beads for 15–20 min. VWF captured by beads was released by 20 mM DTT and detected by a polyclonal anti-VWF antibody under reducing conditions. (B) VWF multimers were incubated with MPB before and after shear exposure (and with and without rADAMTS-13), and MPB-labeled VWF was detected by HRP-streptavidin. (C) Thiol beads captured VWF before (detected in DTT elute [“e” fraction]), but not after (detected in supernatant [“s” fraction]) being exposed to fluid shear stress in the presence of 1% BSA. (D) VWF was exposed to 100 dyn/cm 2 shear stress in the presence (lane 1) or absence (lane 2) of 7 nM of rADAMTS-13, separated on 6% SDS-PAGE, and immunoblotted with a polyclonal VWF antibody. As a control, VWF was subjected to a standard static assay of VWF cleavage at a low–ionic-strength in the presence of 1 mM BaCl 2 and 1.5 M urea for 0 (lane 3) and 16 hrs (lane 4) at 37°C. The figure represents 3-5 separate experiments.

    Techniques Used: Incubation, Labeling, SDS Page

    6) Product Images from "Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy"

    Article Title: Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010873

    Mutation of both BD-N and BD-C2 completely disrupt the binding of CaM to full-length hEAG1. (A) Amino acid sequence of AA 147-209 of EAG1 with the two putative 1-8-14 CaM binding motifs highlighted. (B) CaM overlay blot of the 1-8-14 mutated N-terminus of rEAG1. Detection with HRP-conjugated streptavidin revealed that mutations F151S, A152S (I) completely disrupted binding of biotinylated CaM whereas V164S, L165S (II) and V178S, H179D (III) only caused a reduction in binding. (C–E) FRET measurements in cells transfected with YFP-CaM and Cerulean-labeled full-length hEAG1, mutated in BD-N (F151S, A152S) or in both BD-N and BD-C2 (F151S, A152S, F714S, F717S). Cells were treated with 1 µM ionomycin in the presence of high Ca 2+ . (C) Fluorescence intensity images of Cerulean-hEAG and YFP-CaM after photobleaching and the corresponding FRET efficiency images in pseudo-color (D) Cumulative FRET distribution histograms (probability density function, PDF) and (E) average FRET efficiencies (± SEM) of several cells. Scale bar: 5 µm.
    Figure Legend Snippet: Mutation of both BD-N and BD-C2 completely disrupt the binding of CaM to full-length hEAG1. (A) Amino acid sequence of AA 147-209 of EAG1 with the two putative 1-8-14 CaM binding motifs highlighted. (B) CaM overlay blot of the 1-8-14 mutated N-terminus of rEAG1. Detection with HRP-conjugated streptavidin revealed that mutations F151S, A152S (I) completely disrupted binding of biotinylated CaM whereas V164S, L165S (II) and V178S, H179D (III) only caused a reduction in binding. (C–E) FRET measurements in cells transfected with YFP-CaM and Cerulean-labeled full-length hEAG1, mutated in BD-N (F151S, A152S) or in both BD-N and BD-C2 (F151S, A152S, F714S, F717S). Cells were treated with 1 µM ionomycin in the presence of high Ca 2+ . (C) Fluorescence intensity images of Cerulean-hEAG and YFP-CaM after photobleaching and the corresponding FRET efficiency images in pseudo-color (D) Cumulative FRET distribution histograms (probability density function, PDF) and (E) average FRET efficiencies (± SEM) of several cells. Scale bar: 5 µm.

    Techniques Used: Mutagenesis, Binding Assay, Chick Chorioallantoic Membrane Assay, Sequencing, Transfection, Labeling, Fluorescence

    7) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    8) Product Images from "Dynamic Kisspeptin Receptor Trafficking Modulates Kisspeptin-Mediated Calcium Signaling"

    Article Title: Dynamic Kisspeptin Receptor Trafficking Modulates Kisspeptin-Mediated Calcium Signaling

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2013-1165

    Mature KISS1R is resistant to rapid degradation. HEK-myc-KISS1R cells were pretreated with (A) or without (B) CHX for 10 minutes and then incubated with or without 10 −7 M KP10 for the times indicated. The lysates were analyzed by Western blotting using an anti-myc antibody. A representative blot of 3 independent experiments with similar results is shown. C, HEK-myc-KISS1R cells were biotinylated and incubated in the presence or absence of 10 −7 M KP10 for 4 or 24 hours. The cells were lysed and immunoprecipitated (IP) with an anti-myc antibody. The immunoprecipitant was analyzed by Western blotting (IB) using HRP-streptavidin (top panel) or HRP-anti-myc antibody (bottom panel). A representative blot of 3 independent experiments with similar results is shown.
    Figure Legend Snippet: Mature KISS1R is resistant to rapid degradation. HEK-myc-KISS1R cells were pretreated with (A) or without (B) CHX for 10 minutes and then incubated with or without 10 −7 M KP10 for the times indicated. The lysates were analyzed by Western blotting using an anti-myc antibody. A representative blot of 3 independent experiments with similar results is shown. C, HEK-myc-KISS1R cells were biotinylated and incubated in the presence or absence of 10 −7 M KP10 for 4 or 24 hours. The cells were lysed and immunoprecipitated (IP) with an anti-myc antibody. The immunoprecipitant was analyzed by Western blotting (IB) using HRP-streptavidin (top panel) or HRP-anti-myc antibody (bottom panel). A representative blot of 3 independent experiments with similar results is shown.

    Techniques Used: Incubation, Western Blot, Immunoprecipitation

    9) Product Images from "Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules"

    Article Title: Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.017

    Site-specific biotinylation of the anti-hTfR1 antibody. (A) Schematic representation of conjugation of antibodies randomly biotinylated at lysine residues, the Fab fragment specifically biotinylated at C-terminal cysteines, and the biotinylated EGF. (B) ① disulfide bonds between heavy chains of anti-hTfR1 antibody are reduced by 2-mercaptethanolamine; ② the reduced cysteines were biotinylated with maleimide biotin; and ③ Fc regions of the biotinylated antibody are digested with pepsin. (C) ① Biotinylated anti-hTfR1 antibody; and ② the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody subjected to SDS-PAGE, followed by SyproRuby staining and western blotting, using HRP-conjugated streptavidin. (D) Jurkat cells (1×10 5 cells) were infected with the same amount (1 ng p24/200 µl) of E2 71 eMA or E2 71 mSAH pseudotypes pre-incubated with or without biotinylated anti-hTfR1 antibody or biotinylated Fab fragment of anti-hTfR1 antibody at 200 ng/ml. Transgene (EGFP) expression was analyzed by flow cytometry 3 days post-transduction. The averages and standard derivations of triplicate experiments are shown. (E) The titers (TU/40 µg p24/ml) of VSV-G pseudotype and the E2 71 eMA and mSAH pseudotypes with or without biotin α–hTfR1 Fab conjugation. The titers were calculated by triplicated transduction of Jurkat cells with EGFP-expressing vectors. The averages and standard derivations are shown. (F) Flow cytometric profiles of Jurkat cells (1×10 5 ) transduced with VSV-G, E2 71 eMA or mSAH pseudotypes (1 ng p24/200 µl) conjugated with the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody (10 ng) in the presence or absence of nevirapine (1 µM).
    Figure Legend Snippet: Site-specific biotinylation of the anti-hTfR1 antibody. (A) Schematic representation of conjugation of antibodies randomly biotinylated at lysine residues, the Fab fragment specifically biotinylated at C-terminal cysteines, and the biotinylated EGF. (B) ① disulfide bonds between heavy chains of anti-hTfR1 antibody are reduced by 2-mercaptethanolamine; ② the reduced cysteines were biotinylated with maleimide biotin; and ③ Fc regions of the biotinylated antibody are digested with pepsin. (C) ① Biotinylated anti-hTfR1 antibody; and ② the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody subjected to SDS-PAGE, followed by SyproRuby staining and western blotting, using HRP-conjugated streptavidin. (D) Jurkat cells (1×10 5 cells) were infected with the same amount (1 ng p24/200 µl) of E2 71 eMA or E2 71 mSAH pseudotypes pre-incubated with or without biotinylated anti-hTfR1 antibody or biotinylated Fab fragment of anti-hTfR1 antibody at 200 ng/ml. Transgene (EGFP) expression was analyzed by flow cytometry 3 days post-transduction. The averages and standard derivations of triplicate experiments are shown. (E) The titers (TU/40 µg p24/ml) of VSV-G pseudotype and the E2 71 eMA and mSAH pseudotypes with or without biotin α–hTfR1 Fab conjugation. The titers were calculated by triplicated transduction of Jurkat cells with EGFP-expressing vectors. The averages and standard derivations are shown. (F) Flow cytometric profiles of Jurkat cells (1×10 5 ) transduced with VSV-G, E2 71 eMA or mSAH pseudotypes (1 ng p24/200 µl) conjugated with the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody (10 ng) in the presence or absence of nevirapine (1 µM).

    Techniques Used: Conjugation Assay, SDS Page, Staining, Western Blot, Infection, Incubation, Expressing, Flow Cytometry, Transduction

    10) Product Images from "Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation"

    Article Title: Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.00749

    BirAG can biotinylate itself and proteins in rice protoplasts. (A) Expression and localization of BirA ∗ and BirAG proteins in rice protoplasts. Plasmids used for protoplasts transfection were indicated. (B) Western-blot of biotinylated proteins in rice protoplasts after transient expression of BirA-FLAG, BirA ∗ -FLAG, and BirAG-FLAG, with or without biotin at 50 μM in protoplasts incubation solution. Different plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and different BirA versions, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. p35S, CaMV35S promoter; pUbi, maize ubiquitin promoter; arrow indicate the biotinylated BirAG proteins.
    Figure Legend Snippet: BirAG can biotinylate itself and proteins in rice protoplasts. (A) Expression and localization of BirA ∗ and BirAG proteins in rice protoplasts. Plasmids used for protoplasts transfection were indicated. (B) Western-blot of biotinylated proteins in rice protoplasts after transient expression of BirA-FLAG, BirA ∗ -FLAG, and BirAG-FLAG, with or without biotin at 50 μM in protoplasts incubation solution. Different plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and different BirA versions, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. p35S, CaMV35S promoter; pUbi, maize ubiquitin promoter; arrow indicate the biotinylated BirAG proteins.

    Techniques Used: Expressing, Transfection, Western Blot, Incubation, Staining

    Factors influencing BirAG on biotinylating proteins in rice protoplasts. (A) Biotinylation of proteins increases with the biotin concentration in protoplasts incubation buffer. (B) Influence of ATP on protein biotinylation in rice protoplasts, ATP was added into the protoplasts incubation buffer at the concentration of 10 μM. (C) Influence of protoplasts incubation time and biotin concentrations on protein biotinylation in rice protoplasts. (D) BirAG fused with different target proteins can biotinylated proteins in rice protoplasts. Different treatment and plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and BirAG, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. pUbi, maize ubiquitin promoter; arrows indicate the biotinylated BirAG proteins.
    Figure Legend Snippet: Factors influencing BirAG on biotinylating proteins in rice protoplasts. (A) Biotinylation of proteins increases with the biotin concentration in protoplasts incubation buffer. (B) Influence of ATP on protein biotinylation in rice protoplasts, ATP was added into the protoplasts incubation buffer at the concentration of 10 μM. (C) Influence of protoplasts incubation time and biotin concentrations on protein biotinylation in rice protoplasts. (D) BirAG fused with different target proteins can biotinylated proteins in rice protoplasts. Different treatment and plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and BirAG, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. pUbi, maize ubiquitin promoter; arrows indicate the biotinylated BirAG proteins.

    Techniques Used: Concentration Assay, Incubation, Transfection, Staining

    11) Product Images from "Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads"

    Article Title: Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9579

    Investigation of the interaction of lead indolin-2-one compounds with the Sec residue in the C-terminal active center of TrxR Recombinant rat TrxR (0.9 μM) was incubated with 20 μM of the lead compounds and 200 μM NADPH. At indicated timepoints, an aliquot of enzyme mixture was drawn for TrxR activity measurement by DTNB reduction assay and BIAM labelling at pH 6.5. Top panel: time course of TrxR enzyme activity; bottom panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times. Results presented are representative of three independent experiments.
    Figure Legend Snippet: Investigation of the interaction of lead indolin-2-one compounds with the Sec residue in the C-terminal active center of TrxR Recombinant rat TrxR (0.9 μM) was incubated with 20 μM of the lead compounds and 200 μM NADPH. At indicated timepoints, an aliquot of enzyme mixture was drawn for TrxR activity measurement by DTNB reduction assay and BIAM labelling at pH 6.5. Top panel: time course of TrxR enzyme activity; bottom panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times. Results presented are representative of three independent experiments.

    Techniques Used: Size-exclusion Chromatography, Recombinant, Incubation, Activity Assay, Labeling

    12) Product Images from "Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds *"

    Article Title: Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.233858

    Far Western probing to examine binding of ME0052-Bio to putative target proteins. A , putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx ( 1 ), FolX ( 2 ), z2714 ( 3 ), z3974 ( 4 ), WrbA ( 5 ), SurA ( 6 ), and StcE ( 7 ). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 ( EC ), Y. pseudotuberculosis ( YP ), S. typhimurium ( ST ), P. aeruginosa ( PA ), and S. flexneri ( SF ) or empty vector control (− ve ). Panels show overexpression of target proteins: WrbA ( B ), Tpx ( C ), FolX and FolB ( D ). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio ( E ). The addition of 200 μ m unlabeled ME0052 ( F–H ) strongly affected the binding of the biotinylated probe.
    Figure Legend Snippet: Far Western probing to examine binding of ME0052-Bio to putative target proteins. A , putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx ( 1 ), FolX ( 2 ), z2714 ( 3 ), z3974 ( 4 ), WrbA ( 5 ), SurA ( 6 ), and StcE ( 7 ). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 ( EC ), Y. pseudotuberculosis ( YP ), S. typhimurium ( ST ), P. aeruginosa ( PA ), and S. flexneri ( SF ) or empty vector control (− ve ). Panels show overexpression of target proteins: WrbA ( B ), Tpx ( C ), FolX and FolB ( D ). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio ( E ). The addition of 200 μ m unlabeled ME0052 ( F–H ) strongly affected the binding of the biotinylated probe.

    Techniques Used: Western Blot, Binding Assay, Clone Assay, Lysis, Negative Control, Plasmid Preparation, Over Expression, Mutagenesis

    13) Product Images from "Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens"

    Article Title: Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.9.2739-2748.2003

    Binding of rRsp proteins onto a polystyrene surface. Various concentrations of biotin-labeled rRsp protein or BSA solutions were added to the wells of 96-well polystyrene microtiter plate and incubated at RT for 5 min. After a washing step, HRP-conjugated streptavidin was added to the wells, and the bound rRsp proteins were determined. The absorbance values determined at 450 nm are the means from a representative experiment performed with triplicate samples.
    Figure Legend Snippet: Binding of rRsp proteins onto a polystyrene surface. Various concentrations of biotin-labeled rRsp protein or BSA solutions were added to the wells of 96-well polystyrene microtiter plate and incubated at RT for 5 min. After a washing step, HRP-conjugated streptavidin was added to the wells, and the bound rRsp proteins were determined. The absorbance values determined at 450 nm are the means from a representative experiment performed with triplicate samples.

    Techniques Used: Binding Assay, Labeling, Incubation

    14) Product Images from "TDP-43 stabilises the processing intermediates of mitochondrial transcripts"

    Article Title: TDP-43 stabilises the processing intermediates of mitochondrial transcripts

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06953-y

    TDP-43 associates with a subset of L-strand-encoded mt-tRNAs. ( a ) RNAs immunoprecipitated (IP) from DAP (double affinity purification tag, see Supplementary Fig. 1a )-TDP-43–expressing cells or T-REx 293 cells were stained with SYBR Gold. ( b ) A typical MS/MS spectrum of the oligonucleotide [ADUAGp] 2− that originated from the RNase T1 digest of mt-tRNA Asn (top) or [AAUUtm 5 s 2 UUGp] 2− from that of mt-tRNA Gln (bottom). m/z, mass-to-charge ratio. ( c ) Endogenous TDP-43 was immunoprecipitated with anti-TDP-43 (αTDP-43) and analysed by western blotting (WB) with anti-TDP-43. The immunoprecipitated RNAs were detected by northern blotting (NB) with the probes indicated on the right. Rabbit IgG served as a negative control. ( d ) DAP-TDP-43 complex was immunoprecipitated from DAP-TDP-43-expressing T-REx 293 cells with (+) or without (−) UV crosslinking, separated by SDS-PAGE, and detected by WB with horseradish peroxidase (HRP)-conjugated streptavidin (left). The SDS-PAGE gel bands containing RNA-bound DAP-TDP-43 corresponding to the area indicated as 1 and 2 were excised, and RNAs were extracted from the bands and analysed by NB with RNA-specific probes (right). ( e ) Each DAP-TDP-43 mutant bearing a mutation(s) at residue(s) K136, K140, K145, or F147/L149 was immunoprecipitated with anti-FLAG and analysed by WB with HRP-conjugated streptavidin or by NB with the probes indicated on the right. 5S rRNA served as a loading control for RNA and was detected using SYBR Gold staining. ( f ) Recombinant trigger factor–fused TDP-43 (TF-TDP-43) was mixed with synthesised mt-tRNA Asn or its mutant [Ac-stem(Leu), D-loop(Leu), pAntiCdn(Leu), Var-R(Leu) or T-loop(Leu)] shown in Supplementary Fig. 1f , and analysed by electrophoretic mobility shift assay. tRNAs were detected by NB with probes indicated on the left. ( g ) Top: RNAs were immunoprecipitated from TDP-43–expressing T-REx 293 cells in the absence (NT) or presence of synthetic RNA (sRNA-1, sRNA-2, sRNA-3, sRNA-4, or sRNA-5) and analysed by NB with probes indicated on the right. T-REx 293 cells (TR) served as a negative control. Bottom: Sequences of the synthetic RNAs. Asterisks correspond to the sequence that was replaced in Var-R(Leu).
    Figure Legend Snippet: TDP-43 associates with a subset of L-strand-encoded mt-tRNAs. ( a ) RNAs immunoprecipitated (IP) from DAP (double affinity purification tag, see Supplementary Fig. 1a )-TDP-43–expressing cells or T-REx 293 cells were stained with SYBR Gold. ( b ) A typical MS/MS spectrum of the oligonucleotide [ADUAGp] 2− that originated from the RNase T1 digest of mt-tRNA Asn (top) or [AAUUtm 5 s 2 UUGp] 2− from that of mt-tRNA Gln (bottom). m/z, mass-to-charge ratio. ( c ) Endogenous TDP-43 was immunoprecipitated with anti-TDP-43 (αTDP-43) and analysed by western blotting (WB) with anti-TDP-43. The immunoprecipitated RNAs were detected by northern blotting (NB) with the probes indicated on the right. Rabbit IgG served as a negative control. ( d ) DAP-TDP-43 complex was immunoprecipitated from DAP-TDP-43-expressing T-REx 293 cells with (+) or without (−) UV crosslinking, separated by SDS-PAGE, and detected by WB with horseradish peroxidase (HRP)-conjugated streptavidin (left). The SDS-PAGE gel bands containing RNA-bound DAP-TDP-43 corresponding to the area indicated as 1 and 2 were excised, and RNAs were extracted from the bands and analysed by NB with RNA-specific probes (right). ( e ) Each DAP-TDP-43 mutant bearing a mutation(s) at residue(s) K136, K140, K145, or F147/L149 was immunoprecipitated with anti-FLAG and analysed by WB with HRP-conjugated streptavidin or by NB with the probes indicated on the right. 5S rRNA served as a loading control for RNA and was detected using SYBR Gold staining. ( f ) Recombinant trigger factor–fused TDP-43 (TF-TDP-43) was mixed with synthesised mt-tRNA Asn or its mutant [Ac-stem(Leu), D-loop(Leu), pAntiCdn(Leu), Var-R(Leu) or T-loop(Leu)] shown in Supplementary Fig. 1f , and analysed by electrophoretic mobility shift assay. tRNAs were detected by NB with probes indicated on the left. ( g ) Top: RNAs were immunoprecipitated from TDP-43–expressing T-REx 293 cells in the absence (NT) or presence of synthetic RNA (sRNA-1, sRNA-2, sRNA-3, sRNA-4, or sRNA-5) and analysed by NB with probes indicated on the right. T-REx 293 cells (TR) served as a negative control. Bottom: Sequences of the synthetic RNAs. Asterisks correspond to the sequence that was replaced in Var-R(Leu).

    Techniques Used: Immunoprecipitation, Affinity Purification, Expressing, Staining, Mass Spectrometry, Western Blot, Northern Blot, Negative Control, SDS Page, Mutagenesis, Recombinant, Electrophoretic Mobility Shift Assay, Sequencing

    15) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    16) Product Images from "Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers"

    Article Title: Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers

    Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

    doi: 10.1089/mab.2015.0062

    Immunoblotting and immunoprecipitation assays. (A) Each mAb (2-108 and 4G10) was tested for immunoblotting against lysates of Vero (V), Vero-H (VH), or Vero-mH (VmH) cells under nonreducing and reducing conditions. Anti-mouse HB-EGF pAb M-18 (M18) was used to confirm mouse HB-EGF expression in Vero-mH cells. In immunoblotting, the differences in band pattern of mouse HB-EGF from that of human HB-EGF are due to differences in post-translational processing and modifications. (B) Each mAb (2-108 and 4G10) was tested for immunoblotting against lysates of Vero (V) or Vero-H (VH) cells under nonreducing and reducing conditions at the indicated concentrations. (C) Immunoprecipitation assay. Vero (V), Vero-H (VH), and Vero-mH (VmH) cells were labeled by biotin, and cell lysates were immunoprecipitated with each mAb. The precipitated proteins were analyzed by SDS-PAGE and western blotting using HRP-conjugated streptavidin. HB-EGF, heparin-binding EGF-like growth factor; HRP, horseradish peroxidase; pAb, polyclonal antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
    Figure Legend Snippet: Immunoblotting and immunoprecipitation assays. (A) Each mAb (2-108 and 4G10) was tested for immunoblotting against lysates of Vero (V), Vero-H (VH), or Vero-mH (VmH) cells under nonreducing and reducing conditions. Anti-mouse HB-EGF pAb M-18 (M18) was used to confirm mouse HB-EGF expression in Vero-mH cells. In immunoblotting, the differences in band pattern of mouse HB-EGF from that of human HB-EGF are due to differences in post-translational processing and modifications. (B) Each mAb (2-108 and 4G10) was tested for immunoblotting against lysates of Vero (V) or Vero-H (VH) cells under nonreducing and reducing conditions at the indicated concentrations. (C) Immunoprecipitation assay. Vero (V), Vero-H (VH), and Vero-mH (VmH) cells were labeled by biotin, and cell lysates were immunoprecipitated with each mAb. The precipitated proteins were analyzed by SDS-PAGE and western blotting using HRP-conjugated streptavidin. HB-EGF, heparin-binding EGF-like growth factor; HRP, horseradish peroxidase; pAb, polyclonal antibody; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

    Techniques Used: Immunoprecipitation, Expressing, Labeling, SDS Page, Western Blot, Binding Assay, Polyacrylamide Gel Electrophoresis

    17) Product Images from "IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling"

    Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0273-7

    IGF-1R is preferentially expressed in TMZ-resistant glioma cells. a Cell surface biotinylated proteins, derived from lysed TMZ-sensitive (U251) and TMZ-resistant (UTMZ) glioma cells, were resolved on SDS/PAGE gels, and transferred to PVDF membranes. Proteins were detected by Western blot with HRP-streptavidin (left), and anti-IGF-1R antibody (right). b Visualization of the cell surface of non-fixed, live TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cells with immunofluorescence using fluorescein-conjugated antibodies against IGF-1R (bottom panel) and isotype control (top panel). Magnification 400×. c Expression of IGF-1R in GBM and low-grade gliomas compared with non-tumor tissue. Median values are represented as lines in the scatter plot. * P
    Figure Legend Snippet: IGF-1R is preferentially expressed in TMZ-resistant glioma cells. a Cell surface biotinylated proteins, derived from lysed TMZ-sensitive (U251) and TMZ-resistant (UTMZ) glioma cells, were resolved on SDS/PAGE gels, and transferred to PVDF membranes. Proteins were detected by Western blot with HRP-streptavidin (left), and anti-IGF-1R antibody (right). b Visualization of the cell surface of non-fixed, live TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cells with immunofluorescence using fluorescein-conjugated antibodies against IGF-1R (bottom panel) and isotype control (top panel). Magnification 400×. c Expression of IGF-1R in GBM and low-grade gliomas compared with non-tumor tissue. Median values are represented as lines in the scatter plot. * P

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Immunofluorescence, Expressing

    18) Product Images from "Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity"

    Article Title: Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2011.206

    Amino-Nogo-A resists to hydrogen peroxide (H 2 O 2 ) via interacting with peroxiredoxin 2 (Prdx2). ( a ) Neurons were incubated with 0.2 μ M HIV-1 trans -activating (TAT)-AM for indicated time, and then cells lysates were performed to western blot with anti-Prdx2-SO3 and anti-2-cysteine (Cys) Prdx2 antibodies. β -Actin was selected as a loading control. ( b ) Lysates from neurons treated by H 2 O 2 with or without TAT-AM were performed to western blot with anti-Prdx2-SO3 and anti-2-Cys Prdx2. β -Actin was selected as a loading control. ( c ) Cortical neurons were incubated with 0.2 μ M TAT-GFP or TAT-AM for 2 h, and then were submitted to Ni 2+ pull-down assay. Bound endogenous Prdx2-SO3 was analyzed by immunoblotting with anti-Prdx2-SO3 antibody. ( d ) Glutathione-Sepharose beads coupled with GST or GST-AM were incubated with TAT-HA-Prdx2. Bound Prdx2 was analyzed by immunoblotting with anti-HA antibody. ( e ) Glutathione-Sepharose beads coupled with GST or GST-Prdx2 were incubated with 293FT cell lysate overexpressed with GFP-M1-WT. Bound M1-WT was analyzed by immunoblotting with anti-GFP antibody. The amount of GST or GST-fusion Prdx2 was revealed by naphthol blue black staining. Neurons pretreated with TAT-Prdx2 or TAT-AM, followed by incubation in H 2 O 2 was examined by cell death assay ( f ) and dihydroethidium (DHE) test ( g ) as before. ( h ) Top: neurons transfected with ctrl-small interfering RNA (siRNA) and Prdx2-siRNA for 48 h were blotted with anti-2-Cys Prdx2 antibody. β -Actin was selected as a loading control. Lower: Neurons transfected with ctrl-siRNA and Prdx2-siRNA were pre-treated with TAT-AM followed by incubation in H 2 O 2 , and then cell death assay was calculated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (i) Top: neurons treated with 0.2 μ M TAT-M1-WT, TAT-M1-3CA and TAT-M1-2CA for 2 h were lysed and immunoblotted with anti-HA antibody. The effect of the three proteins on oxidative damaged neurons was checked by cell death assay (lower in i ) and DHE test ( j ). ( k ) In all, 100 ng M9-WT pre-incubated with 1 mM H 2 O 2 or not were labeled with biotin-conjugated iodoacetamide (BIAM) and then probed with horseradish peroxidase (HRP)-streptavidin. ( l ) Working model of amino-Nogo-A resisting to H 2 O 2 . n =4, mean±S.D., one-way analysis of variance (ANOVA), ** P
    Figure Legend Snippet: Amino-Nogo-A resists to hydrogen peroxide (H 2 O 2 ) via interacting with peroxiredoxin 2 (Prdx2). ( a ) Neurons were incubated with 0.2 μ M HIV-1 trans -activating (TAT)-AM for indicated time, and then cells lysates were performed to western blot with anti-Prdx2-SO3 and anti-2-cysteine (Cys) Prdx2 antibodies. β -Actin was selected as a loading control. ( b ) Lysates from neurons treated by H 2 O 2 with or without TAT-AM were performed to western blot with anti-Prdx2-SO3 and anti-2-Cys Prdx2. β -Actin was selected as a loading control. ( c ) Cortical neurons were incubated with 0.2 μ M TAT-GFP or TAT-AM for 2 h, and then were submitted to Ni 2+ pull-down assay. Bound endogenous Prdx2-SO3 was analyzed by immunoblotting with anti-Prdx2-SO3 antibody. ( d ) Glutathione-Sepharose beads coupled with GST or GST-AM were incubated with TAT-HA-Prdx2. Bound Prdx2 was analyzed by immunoblotting with anti-HA antibody. ( e ) Glutathione-Sepharose beads coupled with GST or GST-Prdx2 were incubated with 293FT cell lysate overexpressed with GFP-M1-WT. Bound M1-WT was analyzed by immunoblotting with anti-GFP antibody. The amount of GST or GST-fusion Prdx2 was revealed by naphthol blue black staining. Neurons pretreated with TAT-Prdx2 or TAT-AM, followed by incubation in H 2 O 2 was examined by cell death assay ( f ) and dihydroethidium (DHE) test ( g ) as before. ( h ) Top: neurons transfected with ctrl-small interfering RNA (siRNA) and Prdx2-siRNA for 48 h were blotted with anti-2-Cys Prdx2 antibody. β -Actin was selected as a loading control. Lower: Neurons transfected with ctrl-siRNA and Prdx2-siRNA were pre-treated with TAT-AM followed by incubation in H 2 O 2 , and then cell death assay was calculated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (i) Top: neurons treated with 0.2 μ M TAT-M1-WT, TAT-M1-3CA and TAT-M1-2CA for 2 h were lysed and immunoblotted with anti-HA antibody. The effect of the three proteins on oxidative damaged neurons was checked by cell death assay (lower in i ) and DHE test ( j ). ( k ) In all, 100 ng M9-WT pre-incubated with 1 mM H 2 O 2 or not were labeled with biotin-conjugated iodoacetamide (BIAM) and then probed with horseradish peroxidase (HRP)-streptavidin. ( l ) Working model of amino-Nogo-A resisting to H 2 O 2 . n =4, mean±S.D., one-way analysis of variance (ANOVA), ** P

    Techniques Used: Incubation, Western Blot, Pull Down Assay, Staining, Transfection, Small Interfering RNA, MTT Assay, Labeling

    19) Product Images from "A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene *"

    Article Title: A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.461343

    A , all CA46-R cells are CD22-positive, but the intensity is slightly decreased. Cells were incubated on ice with PE-labeled anti-CD22 antibody for 45 min and analyzed by FACS. MFI , geometric mean of fluorescence intensity. B , the internalization rate is also decreased in CA46-R cells, and it is possibly because of lower CD22 expression. Cells were incubated with Alexa Fluor 647-labeled HA22 at 37 °C for 10 and 30 min and 1, 4, and 24 h. Internalized immunotoxin is shown as the geometric mean fluorescence. C , HA22 cannot inhibit leucine incorporation in CA46-R cells. Cells were treated with HA22 for 20 h. Protein synthesis was measured by incorporation of [ 3 H]leucine. Mean triplicate values are shown. D , HA22 is not able to ADP-ribosylate ( ADP-r ) EF2 in CA46-R cells. Cell lysate (30 μg) was incubated with or without 100 ng of HA22 for 60 min at 25 °C. Samples were analyzed by Western blotting with HRP-conjugated streptavidin to detect biotin-ADP-ribose-EF2. EF2 is shown as a loading control.
    Figure Legend Snippet: A , all CA46-R cells are CD22-positive, but the intensity is slightly decreased. Cells were incubated on ice with PE-labeled anti-CD22 antibody for 45 min and analyzed by FACS. MFI , geometric mean of fluorescence intensity. B , the internalization rate is also decreased in CA46-R cells, and it is possibly because of lower CD22 expression. Cells were incubated with Alexa Fluor 647-labeled HA22 at 37 °C for 10 and 30 min and 1, 4, and 24 h. Internalized immunotoxin is shown as the geometric mean fluorescence. C , HA22 cannot inhibit leucine incorporation in CA46-R cells. Cells were treated with HA22 for 20 h. Protein synthesis was measured by incorporation of [ 3 H]leucine. Mean triplicate values are shown. D , HA22 is not able to ADP-ribosylate ( ADP-r ) EF2 in CA46-R cells. Cell lysate (30 μg) was incubated with or without 100 ng of HA22 for 60 min at 25 °C. Samples were analyzed by Western blotting with HRP-conjugated streptavidin to detect biotin-ADP-ribose-EF2. EF2 is shown as a loading control.

    Techniques Used: Incubation, Labeling, FACS, Fluorescence, Expressing, Western Blot

    20) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    21) Product Images from "Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes"

    Article Title: Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10454

    The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.
    Figure Legend Snippet: The Cx43 region involved in the recruitment of PTEN and Csk C6 glioma cells were incubated with several cell-penetrating peptides containing the indicated sequences of Cx43 fused to biotin for 30 min. ( A ) The SH3 domain binding motif is shown in grey and the tyrosines phosphorylated by c-Src in red. ( B ) After 30 min, the cells were lysed, and pull-down assays were carried out with avidin-conjugated agarose beads. Western blots before (lysates) and after avidin pull-down for c-Src, Csk and PTEN showing the enrichment of PTEN and Csk in the complex obtained with TAT-Cx43-266-283-Biotin and, to a lesser extent, with TAT-Cx43-245-283-Biotin. Str-HRP, HRP-conjugated streptavidin. ( C ) After 30 min, the cells were fixed, and the uptake of peptides bound to biotin was analyzed by fluorescence microscopy. Scale bars: 15 μm.

    Techniques Used: Incubation, Binding Assay, Avidin-Biotin Assay, Western Blot, Fluorescence, Microscopy

    22) Product Images from "Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation"

    Article Title: Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2014.1640

    Treatment with β-ME activates TGase2 in HLE-B3 cells. (A and B) HLE-B3 cells were exposed to β-ME (7.5 mM) for the indicated times. Cells were incubated for 1 h with BP (1 mM), and intracellular TGase2 activity was determined by microtiter plate assay (A) and western blot analysis (B). Streptavidin-HRP (St-Avidin) was used to detect the BP incorporated into the proteins. ** p
    Figure Legend Snippet: Treatment with β-ME activates TGase2 in HLE-B3 cells. (A and B) HLE-B3 cells were exposed to β-ME (7.5 mM) for the indicated times. Cells were incubated for 1 h with BP (1 mM), and intracellular TGase2 activity was determined by microtiter plate assay (A) and western blot analysis (B). Streptavidin-HRP (St-Avidin) was used to detect the BP incorporated into the proteins. ** p

    Techniques Used: Incubation, Activity Assay, Western Blot, Avidin-Biotin Assay

    23) Product Images from "Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens"

    Article Title: Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.9.2739-2748.2003

    Binding of rRsp proteins onto a polystyrene surface. Various concentrations of biotin-labeled rRsp protein or BSA solutions were added to the wells of 96-well polystyrene microtiter plate and incubated at RT for 5 min. After a washing step, HRP-conjugated streptavidin was added to the wells, and the bound rRsp proteins were determined. The absorbance values determined at 450 nm are the means from a representative experiment performed with triplicate samples.
    Figure Legend Snippet: Binding of rRsp proteins onto a polystyrene surface. Various concentrations of biotin-labeled rRsp protein or BSA solutions were added to the wells of 96-well polystyrene microtiter plate and incubated at RT for 5 min. After a washing step, HRP-conjugated streptavidin was added to the wells, and the bound rRsp proteins were determined. The absorbance values determined at 450 nm are the means from a representative experiment performed with triplicate samples.

    Techniques Used: Binding Assay, Labeling, Incubation

    24) Product Images from "IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling"

    Article Title: IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0273-7

    IGF-1R is preferentially expressed in TMZ-resistant glioma cells. a Cell surface biotinylated proteins, derived from lysed TMZ-sensitive (U251) and TMZ-resistant (UTMZ) glioma cells, were resolved on SDS/PAGE gels, and transferred to PVDF membranes. Proteins were detected by Western blot with HRP-streptavidin (left), and anti-IGF-1R antibody (right). b Visualization of the cell surface of non-fixed, live TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cells with immunofluorescence using fluorescein-conjugated antibodies against IGF-1R (bottom panel) and isotype control (top panel). Magnification 400×. c Expression of IGF-1R in GBM and low-grade gliomas compared with non-tumor tissue. Median values are represented as lines in the scatter plot. * P
    Figure Legend Snippet: IGF-1R is preferentially expressed in TMZ-resistant glioma cells. a Cell surface biotinylated proteins, derived from lysed TMZ-sensitive (U251) and TMZ-resistant (UTMZ) glioma cells, were resolved on SDS/PAGE gels, and transferred to PVDF membranes. Proteins were detected by Western blot with HRP-streptavidin (left), and anti-IGF-1R antibody (right). b Visualization of the cell surface of non-fixed, live TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cells with immunofluorescence using fluorescein-conjugated antibodies against IGF-1R (bottom panel) and isotype control (top panel). Magnification 400×. c Expression of IGF-1R in GBM and low-grade gliomas compared with non-tumor tissue. Median values are represented as lines in the scatter plot. * P

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Immunofluorescence, Expressing

    25) Product Images from "Anti-idiotypic Antibodies against BP-IgG Prevent Type XVII Collagen Depletion"

    Article Title: Anti-idiotypic Antibodies against BP-IgG Prevent Type XVII Collagen Depletion

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01669

    Intravenous immunoglobulin (IVIg) contains anti-idiotypic antibodies against anti-type XVII collagen (COL17) IgG. (A) To deplete idiotypic antibodies, anti-COL17 non-collagenous 16A (NC16A) IgG was purified. Next, anti-COL17 NC16A IgG was coupled to a column. IVIg was passed through the column, and then the flow-through fraction (IVIg depleted) and the elution fraction (idiotype) were corrected. (B) To evaluate the depletion efficacy, 96-well microtiter plates were coated with anti-COL17 NC16A IgG and normal human IgG (500 ng/well). The plates were incubated with biotin-conjugated IVIg (1 mg/ml; idiotype sample: 0.1 mg/ml). Finally, the plates were incubated with HRP-conjugated streptavidin. The depletion efficacy was calculated as follows: ( IVIg-depleted OD to anti-COL17 NC16A IgG ) – ( IVIg OD to normal IgG ) ( IVIg OD to anti-COL17 NC16A ) – ( IVIg OD to normal IgG ) × 100 . (C) To calculate the depletion efficacy, the relative OD score of biotin-conjugated IVIg (5 mg/ml) against anti-COL17 NC16A IgG was determined. Using IVIg samples, the following were performed: (D) COL17 NC16A ELISA, (E) COL17-depletion assay, and (F) cell adhesion test. Bovine serum albumin (BSA) and normal human IgG at the same concentrations were used as controls. Data are based on duplicate samples, and each experiment was performed three times, with p
    Figure Legend Snippet: Intravenous immunoglobulin (IVIg) contains anti-idiotypic antibodies against anti-type XVII collagen (COL17) IgG. (A) To deplete idiotypic antibodies, anti-COL17 non-collagenous 16A (NC16A) IgG was purified. Next, anti-COL17 NC16A IgG was coupled to a column. IVIg was passed through the column, and then the flow-through fraction (IVIg depleted) and the elution fraction (idiotype) were corrected. (B) To evaluate the depletion efficacy, 96-well microtiter plates were coated with anti-COL17 NC16A IgG and normal human IgG (500 ng/well). The plates were incubated with biotin-conjugated IVIg (1 mg/ml; idiotype sample: 0.1 mg/ml). Finally, the plates were incubated with HRP-conjugated streptavidin. The depletion efficacy was calculated as follows: ( IVIg-depleted OD to anti-COL17 NC16A IgG ) – ( IVIg OD to normal IgG ) ( IVIg OD to anti-COL17 NC16A ) – ( IVIg OD to normal IgG ) × 100 . (C) To calculate the depletion efficacy, the relative OD score of biotin-conjugated IVIg (5 mg/ml) against anti-COL17 NC16A IgG was determined. Using IVIg samples, the following were performed: (D) COL17 NC16A ELISA, (E) COL17-depletion assay, and (F) cell adhesion test. Bovine serum albumin (BSA) and normal human IgG at the same concentrations were used as controls. Data are based on duplicate samples, and each experiment was performed three times, with p

    Techniques Used: Purification, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Depletion Assay

    26) Product Images from "Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation"

    Article Title: Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2014.1640

    Treatment with β-ME activates TGase2 in HLE-B3 cells. (A and B) HLE-B3 cells were exposed to β-ME (7.5 mM) for the indicated times. Cells were incubated for 1 h with BP (1 mM), and intracellular TGase2 activity was determined by microtiter plate assay (A) and western blot analysis (B). Streptavidin-HRP (St-Avidin) was used to detect the BP incorporated into the proteins. ** p
    Figure Legend Snippet: Treatment with β-ME activates TGase2 in HLE-B3 cells. (A and B) HLE-B3 cells were exposed to β-ME (7.5 mM) for the indicated times. Cells were incubated for 1 h with BP (1 mM), and intracellular TGase2 activity was determined by microtiter plate assay (A) and western blot analysis (B). Streptavidin-HRP (St-Avidin) was used to detect the BP incorporated into the proteins. ** p

    Techniques Used: Incubation, Activity Assay, Western Blot, Avidin-Biotin Assay

    27) Product Images from "Nutrient Isothiocyanates Covalently Modify and Inhibit the Inflammatory Cytokine Macrophage Migration Inhibitory Factor (MIF)"

    Article Title: Nutrient Isothiocyanates Covalently Modify and Inhibit the Inflammatory Cytokine Macrophage Migration Inhibitory Factor (MIF)

    Journal: The Biochemical journal

    doi: 10.1042/BJ20091170

    Proteomics screen for protein targets of covalent ITC modification A. Bio-ITC structure. B. Schematic representation of competition based screen . In cells pretreated with PEITC, ITC target sites are occupied by the natural isothiocyanate (P). After lysis and labeling with Bio-ITC, these specific target sites are unavailable, though nonspecific proteins (N) may be labeled. Detection of Bio-ITC will show specific and non-specific proteins. Proteins detected in the samples prepared from the untreated cells, but absent from the PEITC treated cells represent specific targets of the natural isothiocyanate in intact cells. C. Representative labeling experiment. HeLa cells were left untreated or pretreated with 250μM PEITC to promote complete modification of potential target proteins. Lysates were labeled in vitro with Bio-ITC, and labeled proteins detected on SDS-PAGE with HRP conjugated streptavidin. The predominant target of Bio-ITC modification was a small molecular weight protein of less than 29kD, indicated by double arrows; labeling was completely inhibited by pretreatment of the intact cells with PEITC.
    Figure Legend Snippet: Proteomics screen for protein targets of covalent ITC modification A. Bio-ITC structure. B. Schematic representation of competition based screen . In cells pretreated with PEITC, ITC target sites are occupied by the natural isothiocyanate (P). After lysis and labeling with Bio-ITC, these specific target sites are unavailable, though nonspecific proteins (N) may be labeled. Detection of Bio-ITC will show specific and non-specific proteins. Proteins detected in the samples prepared from the untreated cells, but absent from the PEITC treated cells represent specific targets of the natural isothiocyanate in intact cells. C. Representative labeling experiment. HeLa cells were left untreated or pretreated with 250μM PEITC to promote complete modification of potential target proteins. Lysates were labeled in vitro with Bio-ITC, and labeled proteins detected on SDS-PAGE with HRP conjugated streptavidin. The predominant target of Bio-ITC modification was a small molecular weight protein of less than 29kD, indicated by double arrows; labeling was completely inhibited by pretreatment of the intact cells with PEITC.

    Techniques Used: Modification, Lysis, Labeling, In Vitro, SDS Page, Molecular Weight

    28) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    29) Product Images from "Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties"

    Article Title: Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227727

    Probing requirements to bind a 42kDa cellular target and accumulate high molecular weight polyubiquitinated proteins. (A) Chemical structures of candidate inhibitors. (B) Precleared ES2 cell lysate was incubated with compounds (20 μM) or vehicle (DMSO, 1:100) for 45 min and then with RA190B (5 μM) for 45 min at 4°C. Samples were subjected to SDS-PAGE, transfer to PVDF membrane, and after probing with HRP-streptavidin, binding was detected using chemiluminescence. (C) ES2 cells were treated with compounds (1 μM, 4 hr), lysed and the samples probed with ubiquitin or actin-specific antibody by Western blot.
    Figure Legend Snippet: Probing requirements to bind a 42kDa cellular target and accumulate high molecular weight polyubiquitinated proteins. (A) Chemical structures of candidate inhibitors. (B) Precleared ES2 cell lysate was incubated with compounds (20 μM) or vehicle (DMSO, 1:100) for 45 min and then with RA190B (5 μM) for 45 min at 4°C. Samples were subjected to SDS-PAGE, transfer to PVDF membrane, and after probing with HRP-streptavidin, binding was detected using chemiluminescence. (C) ES2 cells were treated with compounds (1 μM, 4 hr), lysed and the samples probed with ubiquitin or actin-specific antibody by Western blot.

    Techniques Used: Molecular Weight, Incubation, SDS Page, Binding Assay, Western Blot

    Impact of modifications around the RA190 core moiety. (A) Pharmacophore is denoted. R, R1 and R2 are modification sites to potentially alter the molecule’s physical and chemical properties while retaining the mechanism of action. Modifications at the green dots may allow the molecule to orient in one of four possible confirmations of cyclohexanone. (B) Precleared OV2008 cell lysate was incubated with compounds (20 μM) or vehicle (DMSO, 1:100) for 45 min and then with RA190B (10 μM) for 45 min at 4°C. Samples were subjected to SDS-PAGE, transfer to PVDF membrane, and binding of HRP-streptavidin, detected using chemiluminescence.
    Figure Legend Snippet: Impact of modifications around the RA190 core moiety. (A) Pharmacophore is denoted. R, R1 and R2 are modification sites to potentially alter the molecule’s physical and chemical properties while retaining the mechanism of action. Modifications at the green dots may allow the molecule to orient in one of four possible confirmations of cyclohexanone. (B) Precleared OV2008 cell lysate was incubated with compounds (20 μM) or vehicle (DMSO, 1:100) for 45 min and then with RA190B (10 μM) for 45 min at 4°C. Samples were subjected to SDS-PAGE, transfer to PVDF membrane, and binding of HRP-streptavidin, detected using chemiluminescence.

    Techniques Used: Modification, Incubation, SDS Page, Binding Assay

    30) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    31) Product Images from "Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags"

    Article Title: Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113708

    Site-specific biotinylation of proNGF and NGF. A–B ) Western blot for the analysis of the in vitro biotinylation reaction of purified NGF-A4 ( A ) and proNGF-A4 ( B ) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged wt NGF and wt proNGF as negative controls. Streptavidin-HRP is used for detection of biotin. The anti-NGF blot is the loading control. C ) Typical DIC images obtained when performing the differentiation assay in PC12 cells using ∼50 ng/ml of wt NGF, NGF-A4 and biotinylated NGF-A4 (NGF-A4b). Untreated cells are the control. Scale bar: 20 µm.
    Figure Legend Snippet: Site-specific biotinylation of proNGF and NGF. A–B ) Western blot for the analysis of the in vitro biotinylation reaction of purified NGF-A4 ( A ) and proNGF-A4 ( B ) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged wt NGF and wt proNGF as negative controls. Streptavidin-HRP is used for detection of biotin. The anti-NGF blot is the loading control. C ) Typical DIC images obtained when performing the differentiation assay in PC12 cells using ∼50 ng/ml of wt NGF, NGF-A4 and biotinylated NGF-A4 (NGF-A4b). Untreated cells are the control. Scale bar: 20 µm.

    Techniques Used: Western Blot, In Vitro, Purification, Differentiation Assay

    Site-specific biotinylation of TrkA and P75NTR receptors. Western blot for the analysis of the biotinylation reaction in living cells of A1/S6-TrkA-EGFP ( A ) and A1/S6-P75NTR-EGFP constructs ( B ) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged TrkA-EGFP ( A ) and P75NTR-EGFP ( B ) as negative controls, and ACP-TrkA ( A ) as positive control. Streptavidin-HRP is used for detection of biotin. Anti-TrkA ( A ) and anti-P75NTR ( B ) blots are loading controls together with anti-GFP (both panels). The anti-TrkA blot contains an unspecific band running over TrkA, as already shown [4] ; the actual TrkA band in each lane is highlighted by a star. At the bottom of each panel the densitometric analysis of the blot bands is reported. The biotin signal was normalized to the content of GFP (for TrkA-EGFP, A1-TrkA-EGFP, S6-TrkA-EGFP lanes), TrkA (for ACP-TrkA lanes), and P75NTR (for P75NTR-EGFP, A1-P75NTR-EGFP, S6-P75NTR-EGFP lanes), with the higher value normalized to 1. Results reported are mean±sem from 3 (panel A) and 2 (panel B) independent blots.
    Figure Legend Snippet: Site-specific biotinylation of TrkA and P75NTR receptors. Western blot for the analysis of the biotinylation reaction in living cells of A1/S6-TrkA-EGFP ( A ) and A1/S6-P75NTR-EGFP constructs ( B ) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged TrkA-EGFP ( A ) and P75NTR-EGFP ( B ) as negative controls, and ACP-TrkA ( A ) as positive control. Streptavidin-HRP is used for detection of biotin. Anti-TrkA ( A ) and anti-P75NTR ( B ) blots are loading controls together with anti-GFP (both panels). The anti-TrkA blot contains an unspecific band running over TrkA, as already shown [4] ; the actual TrkA band in each lane is highlighted by a star. At the bottom of each panel the densitometric analysis of the blot bands is reported. The biotin signal was normalized to the content of GFP (for TrkA-EGFP, A1-TrkA-EGFP, S6-TrkA-EGFP lanes), TrkA (for ACP-TrkA lanes), and P75NTR (for P75NTR-EGFP, A1-P75NTR-EGFP, S6-P75NTR-EGFP lanes), with the higher value normalized to 1. Results reported are mean±sem from 3 (panel A) and 2 (panel B) independent blots.

    Techniques Used: Western Blot, Construct, Positive Control

    32) Product Images from "A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells"

    Article Title: A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26193

    Characterization of TTGM 5826 ( A ) tTG (0.4 μM) was incubated with the indicated amounts of TTGM 5826 (black circles) or CaCl 2 (white circles) at room temperature for 5 minutes, after which bodipy-labeled GTPγS was added to the reactions. The fluorescence of the bodipy label was measured (ex: 504 nm, em: 520 nm), and the percentage of closed state tTG determined. ( B ) tTG (0.4 μM) was incubated with 10 mM CaCl 2 and the indicated amount of TTGM 5826 for 5 minutes, after which bodipy-labeled GTPγS and 20 mM EDTA were added. The reaction was incubated for an additional 10 minutes and then analyzed as described in ( A ). ( C ) Recombinantly expressed tTG (43 nM) was incubated at room temperature for 15 minutes with 10 mM CaCl 2 , 10 mM DTT, 62.5 μM BPA, and the indicated amounts of NNDC and of TTGM 5826. The proteins were resolved by SDS-PAGE and the extent that BPA was incorporated into NNDC was determined by probing the resulting blot with HRP-conjugated streptavidin. ( D ) Quantification of the data shown in ( C ). Band density was determined using ImageJ, and was calculated with respect to the DMSO control from each experiment. The error bars in panels ( A ), ( B ), and ( D ) represent the SD from three independent experiments. The lowest concentration data point for each series in ( A ) and ( B ) is a control experiment (no TTGM 5826 or CaCl 2 ), and is assigned a low, non-zero value to allow plotting on a logarithmic axis.
    Figure Legend Snippet: Characterization of TTGM 5826 ( A ) tTG (0.4 μM) was incubated with the indicated amounts of TTGM 5826 (black circles) or CaCl 2 (white circles) at room temperature for 5 minutes, after which bodipy-labeled GTPγS was added to the reactions. The fluorescence of the bodipy label was measured (ex: 504 nm, em: 520 nm), and the percentage of closed state tTG determined. ( B ) tTG (0.4 μM) was incubated with 10 mM CaCl 2 and the indicated amount of TTGM 5826 for 5 minutes, after which bodipy-labeled GTPγS and 20 mM EDTA were added. The reaction was incubated for an additional 10 minutes and then analyzed as described in ( A ). ( C ) Recombinantly expressed tTG (43 nM) was incubated at room temperature for 15 minutes with 10 mM CaCl 2 , 10 mM DTT, 62.5 μM BPA, and the indicated amounts of NNDC and of TTGM 5826. The proteins were resolved by SDS-PAGE and the extent that BPA was incorporated into NNDC was determined by probing the resulting blot with HRP-conjugated streptavidin. ( D ) Quantification of the data shown in ( C ). Band density was determined using ImageJ, and was calculated with respect to the DMSO control from each experiment. The error bars in panels ( A ), ( B ), and ( D ) represent the SD from three independent experiments. The lowest concentration data point for each series in ( A ) and ( B ) is a control experiment (no TTGM 5826 or CaCl 2 ), and is assigned a low, non-zero value to allow plotting on a logarithmic axis.

    Techniques Used: Incubation, Labeling, Fluorescence, SDS Page, Concentration Assay

    Characterization of onco-Dbl expressing MEFs ( A ) Lysates from uninduced control MEFs, and MEFs induced to express onco-Dbl by removal of doxycycline from the culturing medium, were analyzed by Western blot using HA, tTG, and vinculin (loading control) antibodies. ( B ) Soft-agar colony formation assays were performed on uninduced MEFs, and on MEFs induced to express onco-Dbl. The cells were cultured for four weeks, and the colonies formed in each condition were counted. ( C ) tTG crosslinking assays were conducted on the same cell lysates used in ( A ). An equal amount of each cell lysate was incubated with 10 mM CaCl 2 , 10 mM DTT, and 62.5 μM BPA for 15 minutes, and then resolved by SDS-PAGE. The resulting blot was probed with HRP-streptavidin, and the band densities were quantified with ImageJ. Error bars in ( B ) and ( C ) represent the SD from three independent experiments.
    Figure Legend Snippet: Characterization of onco-Dbl expressing MEFs ( A ) Lysates from uninduced control MEFs, and MEFs induced to express onco-Dbl by removal of doxycycline from the culturing medium, were analyzed by Western blot using HA, tTG, and vinculin (loading control) antibodies. ( B ) Soft-agar colony formation assays were performed on uninduced MEFs, and on MEFs induced to express onco-Dbl. The cells were cultured for four weeks, and the colonies formed in each condition were counted. ( C ) tTG crosslinking assays were conducted on the same cell lysates used in ( A ). An equal amount of each cell lysate was incubated with 10 mM CaCl 2 , 10 mM DTT, and 62.5 μM BPA for 15 minutes, and then resolved by SDS-PAGE. The resulting blot was probed with HRP-streptavidin, and the band densities were quantified with ImageJ. Error bars in ( B ) and ( C ) represent the SD from three independent experiments.

    Techniques Used: Expressing, Western Blot, Cell Culture, Incubation, SDS Page

    33) Product Images from "New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay *"

    Article Title: New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.529578

    Outline of the SPPLAT protocol. A , structure of the biotin-tyramide proximity labeling reagent. B , principle of the method. The antibody-directed targeting of HRP to a surface protein of interest, followed by brief labeling with biotin-tyramide enables proteins in the immediate vicinity of the target to be biotinylated. These are isolated by incubation of the cell lysate with streptavidin-agarose ( SA ), and elution with reducing agent.
    Figure Legend Snippet: Outline of the SPPLAT protocol. A , structure of the biotin-tyramide proximity labeling reagent. B , principle of the method. The antibody-directed targeting of HRP to a surface protein of interest, followed by brief labeling with biotin-tyramide enables proteins in the immediate vicinity of the target to be biotinylated. These are isolated by incubation of the cell lysate with streptavidin-agarose ( SA ), and elution with reducing agent.

    Techniques Used: Labeling, Isolation, Incubation

    Summary of quantitative SILAC data. A, scatter plot showing the isotope ratios for each protein quantitatively identified in both independent SILAC experiments. The organelle locations of the proteins are indicated. Broken lines represent the median value plus 1 S.D. for each data set. B, proteins chB6 and CDC42 were immunoprecipitated from specifically labeled ( S ) and nonspecifically labeled cells ( NS ), then separately probed with antibodies against chB6 or CDC42 and with HRP-streptavidin to detect biotin labeling as indicated. These samples were run under non-reducing conditions. C , immunofluorescence image showing accumulation of mitochondria underneath the BCR surface cluster. Mitochondria were detected using MitoTracker staining, and BCR was detected by immunofluorescence as described under “Experimental Procedures.” Bar = 5 μm.
    Figure Legend Snippet: Summary of quantitative SILAC data. A, scatter plot showing the isotope ratios for each protein quantitatively identified in both independent SILAC experiments. The organelle locations of the proteins are indicated. Broken lines represent the median value plus 1 S.D. for each data set. B, proteins chB6 and CDC42 were immunoprecipitated from specifically labeled ( S ) and nonspecifically labeled cells ( NS ), then separately probed with antibodies against chB6 or CDC42 and with HRP-streptavidin to detect biotin labeling as indicated. These samples were run under non-reducing conditions. C , immunofluorescence image showing accumulation of mitochondria underneath the BCR surface cluster. Mitochondria were detected using MitoTracker staining, and BCR was detected by immunofluorescence as described under “Experimental Procedures.” Bar = 5 μm.

    Techniques Used: Immunoprecipitation, Labeling, Immunofluorescence, Staining

    34) Product Images from "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells"

    Article Title: A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201112098

    BirA* promiscuously biotinylates endogenous proteins in mammalian cells. HeLa cells were analyzed 24 h after transient transfection with myc-BirA-WT or myc-BirA* (R118G). After transfection, cells were cultured either with or without supplemental biotin (50 µM). (a) By Western blot analysis similar levels of the exogenous BirA (asterisk) are detected in all samples with anti-myc. Biotinylated proteins, including both exogenous BirA (asterisk) and endogenous proteins, were detected with HRP-streptavidin. Enhanced protein biotinylation is observed in the myc-BirA* samples as compared with the WT isoform. This difference is dramatically enhanced by the presence of excess biotin. (b) By fluorescence microscopy the BirA is predominantly nuclear as observed with anti-myc (red). Biotinylated proteins were detected with fluorescently labeled streptavidin (green). Considerable biotinylation is only observed in cells expressing myc-BirA* and supplemented with excess biotin. The biotin signal predominantly colocalizes with myc-BirA*. DNA is labeled with Hoechst (blue). Bar, 4 µm.
    Figure Legend Snippet: BirA* promiscuously biotinylates endogenous proteins in mammalian cells. HeLa cells were analyzed 24 h after transient transfection with myc-BirA-WT or myc-BirA* (R118G). After transfection, cells were cultured either with or without supplemental biotin (50 µM). (a) By Western blot analysis similar levels of the exogenous BirA (asterisk) are detected in all samples with anti-myc. Biotinylated proteins, including both exogenous BirA (asterisk) and endogenous proteins, were detected with HRP-streptavidin. Enhanced protein biotinylation is observed in the myc-BirA* samples as compared with the WT isoform. This difference is dramatically enhanced by the presence of excess biotin. (b) By fluorescence microscopy the BirA is predominantly nuclear as observed with anti-myc (red). Biotinylated proteins were detected with fluorescently labeled streptavidin (green). Considerable biotinylation is only observed in cells expressing myc-BirA* and supplemented with excess biotin. The biotin signal predominantly colocalizes with myc-BirA*. DNA is labeled with Hoechst (blue). Bar, 4 µm.

    Techniques Used: Transfection, Cell Culture, Western Blot, Fluorescence, Microscopy, Labeling, Expressing

    35) Product Images from "Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei"

    Article Title: Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-04-0219

    Characterization of the Myc-BirA*-TbPLK cell line. (A) Cells carrying doxycycline-inducible Myc-BirA*-TbPLK were treated with 20 ng/ml doxycycline (+) or a vehicle control (–), and then their lysates were probed with antibodies against TbPLK and Myc. Wild-type cells (control) were included as a control. (B) Myc-BirA*-TbPLK cells were treated (+) with 20 ng/ml doxycycline (DOX), biotin, or vehicle control (–), and then their lysates were probed with HRP-streptavidin to detect biotinylation. (C) Myc-BirA*-TbPLK cells were treated (+DOX) with doxycycline or a vehicle control (Control) and labeled with antibodies against the FAZ (FAZ; red) and either anti-Myc (Myc-BirA*-TbPLK; green) or Dylight 488–conjugated streptavidin (StrepAvidin; green), as well as with DAPI to label DNA (DNA; blue). (D) Myc-BirA*-TbPLK cells were incubated with biotin and either doxycycline (+) or a vehicle control (–), followed by lysis and incubation with streptavidin beads. The beads were washed, and 10% of the bound material was eluted and run on an SDS–PAGE gel, which was then imaged using SYPRO ruby.
    Figure Legend Snippet: Characterization of the Myc-BirA*-TbPLK cell line. (A) Cells carrying doxycycline-inducible Myc-BirA*-TbPLK were treated with 20 ng/ml doxycycline (+) or a vehicle control (–), and then their lysates were probed with antibodies against TbPLK and Myc. Wild-type cells (control) were included as a control. (B) Myc-BirA*-TbPLK cells were treated (+) with 20 ng/ml doxycycline (DOX), biotin, or vehicle control (–), and then their lysates were probed with HRP-streptavidin to detect biotinylation. (C) Myc-BirA*-TbPLK cells were treated (+DOX) with doxycycline or a vehicle control (Control) and labeled with antibodies against the FAZ (FAZ; red) and either anti-Myc (Myc-BirA*-TbPLK; green) or Dylight 488–conjugated streptavidin (StrepAvidin; green), as well as with DAPI to label DNA (DNA; blue). (D) Myc-BirA*-TbPLK cells were incubated with biotin and either doxycycline (+) or a vehicle control (–), followed by lysis and incubation with streptavidin beads. The beads were washed, and 10% of the bound material was eluted and run on an SDS–PAGE gel, which was then imaged using SYPRO ruby.

    Techniques Used: Labeling, Incubation, Lysis, SDS Page

    36) Product Images from "TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2"

    Article Title: TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1119296109

    TG2 is a TBK1-dependent antiapoptotic factor. ( A ) In vivo transamidation activity was determined in WT and Tbk1 −/− MEFs metabolically labeled with BP and stimulated with TNF for the indicated times. Cell extracts were prepared, and biotin-conjugated proteins were detected by immunoblotting using anti–streptavidin-HRP (Pierce). ( B ) Induction of Tg gene expression was determined by qRT-PCR analysis of mRNAs from WT and Tbk1 −/− MEFs stimulated with TNF for the indicated times. ( C ) Endogenous TG2 protein amounts were determined by immunoblotting in extracts from Tbk1 −/− MEFs expressing RelA(E) ( Upper ), PAI-2 ( Lower ), or an “empty” retrovirus (mock) and treated with TNF for the indicated times (ns, nonspecific). ( D ) Coimmunoprecipitation of PAI-2 with TG2 was examined in MEFs stably expressing HA-PAI-2 and human TG2 that were left untreated or were stimulated with TNF for 8 h. Control immunoprecipitations were performed with nonimmune IgGs. ( E ) In vitro transamidation assay was performed with TG2 or control immunoprecipitates prepared from untreated or TNF-stimulated Tbk1 −/− MEFs expressing TG2 and untagged PAI-2 or with recombinant proteins (rec) using HA-procaspase-3 as a substrate. ( F ) Wt MEFs were treated with TNF (20 h) with or without the transglutaminase-specific inhibitor KCC009 (0.5 mM). Caspase-3 activation, PARP cleavage, and protein expression were determined by immunoblotting. ( G ) WT and Tg2 −/− primary MEFs prepared from littermate embryos were left untreated or treated with TNF alone (20 h) or TNF plus CHX (6 h). Caspase-3 activation and apoptosis were determined as detailed earlier. Genotyping ( Lower ). ( H ) The extent of cell death in WT and Tg2 −/− MEFs that were stimulated for 3 h with TNF plus CHX was quantified by staining with PI.
    Figure Legend Snippet: TG2 is a TBK1-dependent antiapoptotic factor. ( A ) In vivo transamidation activity was determined in WT and Tbk1 −/− MEFs metabolically labeled with BP and stimulated with TNF for the indicated times. Cell extracts were prepared, and biotin-conjugated proteins were detected by immunoblotting using anti–streptavidin-HRP (Pierce). ( B ) Induction of Tg gene expression was determined by qRT-PCR analysis of mRNAs from WT and Tbk1 −/− MEFs stimulated with TNF for the indicated times. ( C ) Endogenous TG2 protein amounts were determined by immunoblotting in extracts from Tbk1 −/− MEFs expressing RelA(E) ( Upper ), PAI-2 ( Lower ), or an “empty” retrovirus (mock) and treated with TNF for the indicated times (ns, nonspecific). ( D ) Coimmunoprecipitation of PAI-2 with TG2 was examined in MEFs stably expressing HA-PAI-2 and human TG2 that were left untreated or were stimulated with TNF for 8 h. Control immunoprecipitations were performed with nonimmune IgGs. ( E ) In vitro transamidation assay was performed with TG2 or control immunoprecipitates prepared from untreated or TNF-stimulated Tbk1 −/− MEFs expressing TG2 and untagged PAI-2 or with recombinant proteins (rec) using HA-procaspase-3 as a substrate. ( F ) Wt MEFs were treated with TNF (20 h) with or without the transglutaminase-specific inhibitor KCC009 (0.5 mM). Caspase-3 activation, PARP cleavage, and protein expression were determined by immunoblotting. ( G ) WT and Tg2 −/− primary MEFs prepared from littermate embryos were left untreated or treated with TNF alone (20 h) or TNF plus CHX (6 h). Caspase-3 activation and apoptosis were determined as detailed earlier. Genotyping ( Lower ). ( H ) The extent of cell death in WT and Tg2 −/− MEFs that were stimulated for 3 h with TNF plus CHX was quantified by staining with PI.

    Techniques Used: In Vivo, Activity Assay, Metabolic Labelling, Labeling, Expressing, Quantitative RT-PCR, Stable Transfection, In Vitro, Recombinant, Activation Assay, Staining

    37) Product Images from "An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations"

    Article Title: An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0096-z

    Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin
    Figure Legend Snippet: Reactivity of ERC6 to human EGFR and cotinine. a Cetuximab, anti-cotinine-IgG, and ERC6 were added to wells of a microtiter plate coated with human EGFR (■) or cotinine (□). The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. b Cetuximab, anti-cotinine-IgG, and ERC6 were added to different wells of a microtiter plate coated with human EGFR (■). After addition of Cot-Biotin-Cot peptide, wells were probed with HRP-conjugated streptavidin. The background signal was measured in control wells that were coated with BSA (□). Absorbance at 650 nm was measured. The results are shown as the mean ± SD acquired from experiments conducted in triplicate. EGFR epidermal growth factor receptor, HRP horseradish peroxidase, BSA bovine serum albumin

    Techniques Used:

    38) Product Images from "Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation"

    Article Title: Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    Journal: Molecular Biosystems

    doi: 10.1039/c005279e

    In vivo biotinylation and multi tags strongly improve immobilization of sdAbs A) Serial dilutions of pure anti-Nef sdAb biotinylated in vivo (●), in vitro (■) or unbiotinylated (▲) were coated on streptavidin plate and incubated with Nef at 5 nM. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. B) Protein G bead were coated with 1 μg/ml of 9E10 mAb. Serial dilutions of pure sdAbs with three (●), one (■) or no (▲) myc tag were incubated with the bead, followed by biotinylated Nef at 5 nM. The captured antigen was detected with a HRP-conjugated streptavidin. Standard deviation represents two experiments performed in triplicates.
    Figure Legend Snippet: In vivo biotinylation and multi tags strongly improve immobilization of sdAbs A) Serial dilutions of pure anti-Nef sdAb biotinylated in vivo (●), in vitro (■) or unbiotinylated (▲) were coated on streptavidin plate and incubated with Nef at 5 nM. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. B) Protein G bead were coated with 1 μg/ml of 9E10 mAb. Serial dilutions of pure sdAbs with three (●), one (■) or no (▲) myc tag were incubated with the bead, followed by biotinylated Nef at 5 nM. The captured antigen was detected with a HRP-conjugated streptavidin. Standard deviation represents two experiments performed in triplicates.

    Techniques Used: In Vivo, In Vitro, Incubation, Standard Deviation

    Functional sdAbs are efficiently produced in the cytoplasm of E. coli A) MC38-CEA (■, ●) or MC38 (▲) cells were incubated with serial dilutions of anti-CEA sdAb produced in cytoplasm (■) or periplasm (●) of E. coli . Captured antibodies were detected by a mouse anti-6his mAb followed by a goat against mouse FITC-conjugated mAb. Cells were analyzed by flow cytometry assay on FACScalibur. B) Biotinylated Nef antigen (5 nM) coated on streptavidin-plate was incubated with serial dilution of anti-Nef sdAb produced in the cytoplasm (■) or periplasm (●) of E. coli or anti-CEA sdAb produced in the cytoplasm of E. coli (▲). Captured antibodies were detected by a mouse anti c-myc mAb followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.
    Figure Legend Snippet: Functional sdAbs are efficiently produced in the cytoplasm of E. coli A) MC38-CEA (■, ●) or MC38 (▲) cells were incubated with serial dilutions of anti-CEA sdAb produced in cytoplasm (■) or periplasm (●) of E. coli . Captured antibodies were detected by a mouse anti-6his mAb followed by a goat against mouse FITC-conjugated mAb. Cells were analyzed by flow cytometry assay on FACScalibur. B) Biotinylated Nef antigen (5 nM) coated on streptavidin-plate was incubated with serial dilution of anti-Nef sdAb produced in the cytoplasm (■) or periplasm (●) of E. coli or anti-CEA sdAb produced in the cytoplasm of E. coli (▲). Captured antibodies were detected by a mouse anti c-myc mAb followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

    Techniques Used: Functional Assay, Produced, Incubation, Flow Cytometry, Cytometry, Serial Dilution, Standard Deviation

    Bacterial lysates are a good source of capture antibody Streptavidin beads were coated with sdAb against Nef biotinylated in vivo pure (●) (1 μg/ml) or in bacterial lysate (▲) (50 nl/wells) or sdAb against Nef unbiotinylated in bacterial lysate (▼) and incubated with serial dilution of Nef. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.
    Figure Legend Snippet: Bacterial lysates are a good source of capture antibody Streptavidin beads were coated with sdAb against Nef biotinylated in vivo pure (●) (1 μg/ml) or in bacterial lysate (▲) (50 nl/wells) or sdAb against Nef unbiotinylated in bacterial lysate (▼) and incubated with serial dilution of Nef. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

    Techniques Used: In Vivo, Incubation, Serial Dilution, Standard Deviation

    In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with 9E10 (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or PBS (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.
    Figure Legend Snippet: In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with 9E10 (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or PBS (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

    Techniques Used: In Vivo, Incubation, Standard Deviation

    39) Product Images from "A Bead Aggregation Assay for Detection of Low-Affinity Protein-Protein Interactions Reveals Interactions between N-Terminal Domains of Inositol 1,4,5-Trisphosphate Receptors"

    Article Title: A Bead Aggregation Assay for Detection of Low-Affinity Protein-Protein Interactions Reveals Interactions between N-Terminal Domains of Inositol 1,4,5-Trisphosphate Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060609

    The NT of IP 3 R1 causes aggregation of beads. (A) DIC images of beads prepared as indicated. (B) Summary results show means ± SEM, n = 3. * P = 0.012 relative to SVA beads. (C) Western blot, probed using HRP-conjugated streptavidin, shows equivalent fractions of the input (20 pmol of native or denatured NT-biotin, lane 1), the supernatant (lane 2), wash (lane 3) and NT-beads treated (85°C, 10 min) to release bound protein (lane 4). Lane 5 shows the biotinylated protein ladder. The ∼70-kDa bands correspond to NT-biotin. The blot is representative of two similar analyses. (D) DIC images of NT-biotin-coated SVA-beads with or without 10 µM IP 3 . (E) Summary results show means ± SEM, n = 5. P = 0.40. Scale bars (A and D) represent 20 µm.
    Figure Legend Snippet: The NT of IP 3 R1 causes aggregation of beads. (A) DIC images of beads prepared as indicated. (B) Summary results show means ± SEM, n = 3. * P = 0.012 relative to SVA beads. (C) Western blot, probed using HRP-conjugated streptavidin, shows equivalent fractions of the input (20 pmol of native or denatured NT-biotin, lane 1), the supernatant (lane 2), wash (lane 3) and NT-beads treated (85°C, 10 min) to release bound protein (lane 4). Lane 5 shows the biotinylated protein ladder. The ∼70-kDa bands correspond to NT-biotin. The blot is representative of two similar analyses. (D) DIC images of NT-biotin-coated SVA-beads with or without 10 µM IP 3 . (E) Summary results show means ± SEM, n = 5. P = 0.40. Scale bars (A and D) represent 20 µm.

    Techniques Used: Western Blot

    The SD of IP 3 R1 causes aggregation of beads. (A) Silver-stained gel using equivalent amounts of material shows the proteins present in the incubation used to prepare SVA beads (input), the unbound protein (supernatant), the washings from the beads (wash), and the proteins eluted from the beads (eluate, 85°C for 10 min). (B) Summary results show the percentages of protein immobilized on the beads for NT, IBC and SD. (C) Equilibrium competition binding to NT-biotin and IBC-biotin with 3 H-IP 3 (0.75 nM). (D) Functional protein quantified from the amount of 3 H-IP 3 (mol) bound per mol of protein. Results (C and D) are means ± SEM, n = 3. (E) DIC images of the indicated beads. Scale bar = 20 µm. (F) Summary results (means ± SEM, from 3 independent experiments each with 5 fields). * P = 0.041 relative to NT-beads. (G) Western blot, probed using HRP-conjugated streptavidin, shows purified samples of SD-biotin (lane 1; ∼27 kDa), IBC-biotin (lane 2; ∼44 kDa) and NT-biotin (lane 3; ∼70 kDa). Lane 1 is from a separate blot. The protein preparations did not contain detectable amounts of residual GST-tagged SD, IBC or NT fragments which have predicted sizes of 54 kDa, 71 kDa and 96 kDa, respectively. (H) The SD (left) or NT (right) interact and thereby cause beads to aggregate, whereas beads coated with IBC (centre) do not aggregate.
    Figure Legend Snippet: The SD of IP 3 R1 causes aggregation of beads. (A) Silver-stained gel using equivalent amounts of material shows the proteins present in the incubation used to prepare SVA beads (input), the unbound protein (supernatant), the washings from the beads (wash), and the proteins eluted from the beads (eluate, 85°C for 10 min). (B) Summary results show the percentages of protein immobilized on the beads for NT, IBC and SD. (C) Equilibrium competition binding to NT-biotin and IBC-biotin with 3 H-IP 3 (0.75 nM). (D) Functional protein quantified from the amount of 3 H-IP 3 (mol) bound per mol of protein. Results (C and D) are means ± SEM, n = 3. (E) DIC images of the indicated beads. Scale bar = 20 µm. (F) Summary results (means ± SEM, from 3 independent experiments each with 5 fields). * P = 0.041 relative to NT-beads. (G) Western blot, probed using HRP-conjugated streptavidin, shows purified samples of SD-biotin (lane 1; ∼27 kDa), IBC-biotin (lane 2; ∼44 kDa) and NT-biotin (lane 3; ∼70 kDa). Lane 1 is from a separate blot. The protein preparations did not contain detectable amounts of residual GST-tagged SD, IBC or NT fragments which have predicted sizes of 54 kDa, 71 kDa and 96 kDa, respectively. (H) The SD (left) or NT (right) interact and thereby cause beads to aggregate, whereas beads coated with IBC (centre) do not aggregate.

    Techniques Used: Staining, Incubation, Binding Assay, Functional Assay, Western Blot, Purification

    40) Product Images from "Dermatopontin Interacts with Fibronectin, Promotes Fibronectin Fibril Formation, and Enhances Cell Adhesion *"

    Article Title: Dermatopontin Interacts with Fibronectin, Promotes Fibronectin Fibril Formation, and Enhances Cell Adhesion *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.179762

    Modification of interdomain interactions of Fn by DP. A , enhancement of the interaction by DP. Recombinant Fn domains shown on the x axis were immobilized in wells at 20 μg/ml. A biotinylated 30-kDa fragment of Fn was added at 5 μg/ml in the presence or absence of 10 μg/ml DP. B , inhibition of the interaction by DP. Recombinant III 12–14 was immobilized at 1 μg/ml, and biotinylated III 2–3 was added at 0.2 μg/ml in the presence or absence of 2 μg/ml DP. The bound 30-kDa fragment and the III 2–3 were probed by a streptavidin-conjugated HRP as described under “Experimental Procedures.” *, p
    Figure Legend Snippet: Modification of interdomain interactions of Fn by DP. A , enhancement of the interaction by DP. Recombinant Fn domains shown on the x axis were immobilized in wells at 20 μg/ml. A biotinylated 30-kDa fragment of Fn was added at 5 μg/ml in the presence or absence of 10 μg/ml DP. B , inhibition of the interaction by DP. Recombinant III 12–14 was immobilized at 1 μg/ml, and biotinylated III 2–3 was added at 0.2 μg/ml in the presence or absence of 2 μg/ml DP. The bound 30-kDa fragment and the III 2–3 were probed by a streptavidin-conjugated HRP as described under “Experimental Procedures.” *, p

    Techniques Used: Modification, Recombinant, Inhibition

    41) Product Images from "Precursor and mature NGF live tracking: one versus many at a time in the axons"

    Article Title: Precursor and mature NGF live tracking: one versus many at a time in the axons

    Journal: Scientific Reports

    doi: 10.1038/srep20272

    Schematic overview of production of fluorescent NTs and characterization of fluo-NGF biological activity. ( A ) Cartoon depicting the two-steps labelling strategy. Structure of human NGF (blue ribbon, PDB 1SG1) with overlaid, in grey, the pro-peptide domain 22 ( Left) ; the tag sequence inserted at the C-terminal position of proNGF is depicted in red ( Middle ); the complete structural formula of Alexa488-maleimide-phosphopantetheinyl is added, highlighted in green ( Right ). ( B ) Scheme of proNGF sequence with highlighted tag insertion site. ( C ) Amino acidic sequence of the four screened tags, with the serine residue covalently conjugated to the fluorophore highlighted in red. ( D ) Purification yields (mg of product per litre of bacterial culture) of tagged proNGF-tag (gray) and NGF-tag (blue), compared to wt (pro)NGF. ( E ) Western blot analysis of the biotinylation reaction of purified NGF-YBBR and NGF-A4 using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged wt-NGF as negative control. Streptavidin-HRP is used for biotin detection. The anti-NGF blot (NGF and proNGF lines) is the loading control. ( F ) The HPLC chromatogram of NGF-YBBR, incubated with CoA-Alexa488 substrate in the presence and absence of Sfp-synthase, showing the different retention times of fluorescent and non-fluorescent NTs. Absorbance curves at 280 and 490 nm are reported. ( G ) Typical DIC images of PC12 differentiation assay using equimolar amounts of wt-NGF and fluo-NGF. Untreated cells are the control. Scale bars represent 20 μm. ( H ) Western blot analysis of phosphorylated Akt (pAkt), phosphorylated Erk1/2 (pErk1/2) and phosphorylated PLCγ (pPLCγ) protein levels in PC12 cells in response to wt NGF and fluo-NGF, compared to the same obtained for untreated cells (No NGF); the signal of the total corresponding signalling effectors is the loading control. ( I ) Hierarchical clustering tree of samples, corresponding to the different experimental points (for each neurotrophin type four individual PC12 cells administrations were performed). The trees show the gene expression similarity between samples. The x-axis indicates the distance between samples. Euclidean distance is the chosen metric, with average linkage clustering, using all normalized Log2 data.
    Figure Legend Snippet: Schematic overview of production of fluorescent NTs and characterization of fluo-NGF biological activity. ( A ) Cartoon depicting the two-steps labelling strategy. Structure of human NGF (blue ribbon, PDB 1SG1) with overlaid, in grey, the pro-peptide domain 22 ( Left) ; the tag sequence inserted at the C-terminal position of proNGF is depicted in red ( Middle ); the complete structural formula of Alexa488-maleimide-phosphopantetheinyl is added, highlighted in green ( Right ). ( B ) Scheme of proNGF sequence with highlighted tag insertion site. ( C ) Amino acidic sequence of the four screened tags, with the serine residue covalently conjugated to the fluorophore highlighted in red. ( D ) Purification yields (mg of product per litre of bacterial culture) of tagged proNGF-tag (gray) and NGF-tag (blue), compared to wt (pro)NGF. ( E ) Western blot analysis of the biotinylation reaction of purified NGF-YBBR and NGF-A4 using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged wt-NGF as negative control. Streptavidin-HRP is used for biotin detection. The anti-NGF blot (NGF and proNGF lines) is the loading control. ( F ) The HPLC chromatogram of NGF-YBBR, incubated with CoA-Alexa488 substrate in the presence and absence of Sfp-synthase, showing the different retention times of fluorescent and non-fluorescent NTs. Absorbance curves at 280 and 490 nm are reported. ( G ) Typical DIC images of PC12 differentiation assay using equimolar amounts of wt-NGF and fluo-NGF. Untreated cells are the control. Scale bars represent 20 μm. ( H ) Western blot analysis of phosphorylated Akt (pAkt), phosphorylated Erk1/2 (pErk1/2) and phosphorylated PLCγ (pPLCγ) protein levels in PC12 cells in response to wt NGF and fluo-NGF, compared to the same obtained for untreated cells (No NGF); the signal of the total corresponding signalling effectors is the loading control. ( I ) Hierarchical clustering tree of samples, corresponding to the different experimental points (for each neurotrophin type four individual PC12 cells administrations were performed). The trees show the gene expression similarity between samples. The x-axis indicates the distance between samples. Euclidean distance is the chosen metric, with average linkage clustering, using all normalized Log2 data.

    Techniques Used: Activity Assay, Sequencing, Purification, Western Blot, Negative Control, High Performance Liquid Chromatography, Incubation, Differentiation Assay, Expressing

    Related Articles

    Construct:

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)
    Article Snippet: For detection of biotinylated proteins, streptavidin-HRP (#21126, Pierce) was utilized. .. For the assessment of cellular LRRC32 expression in the context of the signal peptide deletion constructs, an identical protocol as above was used except that cells were lysed using the M-PER Mammalian Protein Extraction Reagent (#78503, Pierce).

    Real-time Polymerase Chain Reaction:

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Paragraph title: Western blot, IHC/IF, VVA pulldown and real-time PCR ... Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used.

    Sandwich ELISA:

    Article Title: Substrate Reduction Augments the Efficacy of Enzyme Therapy in a Mouse Model of Fabry Disease
    Article Snippet: Quantitation of urine uromodulin A sandwich ELISA was developed using commercially available reagents to quantitate uromodulin levels in the urine. .. Following incubation with streptavidin-HRP (Cat# 21127, Thermo Scientific, Rockland, IL) signal was detected using TMB HRP substrate (BioFX Laboratories, Inc., Owings Mills, MD).

    Incubation:

    Article Title: A dual-targeting PDGFR?/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo
    Article Snippet: Concurrently, in a separate 96 well plate (Costar 3357) a 1:1 dilution of supernatant, containing either Fab or scFv and biotinylated VEGF-A (ZymoGenetics) in 1% BSA/PBST at 20 ng/mL was made and incubated for 1 hr at RT. .. After washing, a 1:4,000 dilution of Streptavidin-HRP (#21124, Pierce) in 1% BSA/PBST was added for one hour at RT.

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: .. Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce). .. For development of the peroxidase activity, 3,3′-diaminobenzidine (DAB) chromogen was used.

    Article Title: Substrate Reduction Augments the Efficacy of Enzyme Therapy in a Mouse Model of Fabry Disease
    Article Snippet: .. Following incubation with streptavidin-HRP (Cat# 21127, Thermo Scientific, Rockland, IL) signal was detected using TMB HRP substrate (BioFX Laboratories, Inc., Owings Mills, MD). .. Histopathology At the termination of the study, mice were killed by carbon dioxide asphyxiation.

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: .. Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845). .. Sections were dehydrated and coverslipped with Cytoseal (Thermo Scientific 8312-4).

    Article Title: Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases
    Article Snippet: Streptavidin agarose was then used to pull down bound proteins by incubation the agarose with the treated cell lysate overnight. .. Western Blot was performed as we reported previously using streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2 (Cell Signaling Technology, #4290, Boston, MA) antibodies.

    Article Title: A dual-targeting PDGFR?/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo
    Article Snippet: Next PDGFRβ was added at 0.25 µg/mL in 2% BSA (#160069 MB Biomedicals)/PBST and incubated for one hour at RT. .. Plates were washed with PBST followed by the addition of a 1:3,000 dilution of Streptavidin-HRP (#21124, Pierce) in 2% BSA/PBST for one hour at room temperature.

    Article Title: Mast cells are required for experimental oral allergen-induced diarrhea
    Article Snippet: .. After 30 minutes of incubation and one wash, streptavidin-HRP (1:20,000 dilution) (Immuno Pure Streptavidin Horseradish Peroxidase Conjugated II, 21126; Pierce Chemical Co.) was added. ..

    Article Title: Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer
    Article Snippet: Briefly, 100 μL of the mouse anti-human TF antibody, hTF1 (5 μg mL−1 in PBS), was coated onto 96-well ELISA plates (Nunc No. 439454) by overnight incubation at 4 °C. .. Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well.

    Activity Assay:

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: The endogenous peroxidase activity of the tissues and unspecific epitopes was blocked with “peroxidase blocking reagent” during 5 min and “protein blocking reagent” during 30 min, respectively. .. Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce).

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: For myelin oligodendrocyte glycoprotein (MOG) immunohistochemistry, mounted cryosections were rinsed, delipidated with 100% ethanol for 10 min, and then incubated in 3% H2 O2 to eliminate endogenous peroxidase activity. .. Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845).

    Expressing:

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)
    Article Snippet: For detection of biotinylated proteins, streptavidin-HRP (#21126, Pierce) was utilized. .. For the assessment of cellular LRRC32 expression in the context of the signal peptide deletion constructs, an identical protocol as above was used except that cells were lysed using the M-PER Mammalian Protein Extraction Reagent (#78503, Pierce).

    Western Blot:

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Paragraph title: Western blot, IHC/IF, VVA pulldown and real-time PCR ... Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used.

    Article Title: Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases
    Article Snippet: .. Western Blot was performed as we reported previously using streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2 (Cell Signaling Technology, #4290, Boston, MA) antibodies. .. Molecular dockings The FlexX-Dock module of Sybyl version 7.1 software (Tripos Associates Inc., St. Louis, MO) was used for molecular docking study, which is used for predicting the ligand-receptor interaction modes and for hit identification by structure-based virtual screening.

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)
    Article Snippet: Paragraph title: Biotinylation of cell surface proteins, Immunoprecipitation and Western Blotting ... For detection of biotinylated proteins, streptavidin-HRP (#21126, Pierce) was utilized.

    Blocking Assay:

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: The endogenous peroxidase activity of the tissues and unspecific epitopes was blocked with “peroxidase blocking reagent” during 5 min and “protein blocking reagent” during 30 min, respectively. .. Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce).

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: After permeabilization, slides were first incubated in M.O.M. mouse Ig blocking reagent for 1 h followed by a 30 min incubation with CC1 antibody in M.O.M. diluent and then standard blocking solution and other primary antibodies. .. Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845).

    Immunohistochemistry:

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: All the reagents used were from NovoLink Polymer Detection System Kit for IHC (Novocastra, Millipore). .. Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce).

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. .. For immunohistochemistry, antibodies for Tn Antigen (MA180055; Thermo Fisher Scientific, Grand Island, NY, USA), STn Antigen (ab115957; Abcam), insulin (8138; CST, Beverly, MA, USA) and VVA-fluorescein (FL-1231; Vector Laboratories) were used at a dilution of 1:100 .

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: For myelin oligodendrocyte glycoprotein (MOG) immunohistochemistry, mounted cryosections were rinsed, delipidated with 100% ethanol for 10 min, and then incubated in 3% H2 O2 to eliminate endogenous peroxidase activity. .. Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845).

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. .. For immunohistochemistry, antibodies for Tn Antigen ( ; Thermo Fisher Scientific, Grand Island, NY, USA), STn Antigen (ab115957; Abcam), insulin (8138; CST, Beverly, MA, USA) and VVA-fluorescein (FL-1231; Vector Laboratories) were used at a dilution of 1:100 .

    Recombinant:

    Article Title: Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer
    Article Snippet: Serial dilutions of a recombinant soluble TF1–219 (sTF) standard were added to the plates in duplicate. .. Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well.

    Immunohistochemistry-IF:

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Paragraph title: Western blot, IHC/IF, VVA pulldown and real-time PCR ... Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used.

    Pull Down Assay:

    Article Title: Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases
    Article Snippet: Biological evaluation The pull down assay was carried out using the Immunoprecipitation Kit (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions with minor modifications. .. Western Blot was performed as we reported previously using streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2 (Cell Signaling Technology, #4290, Boston, MA) antibodies.

    Microscopy:

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce). .. Finally, slides were mounted in HiMo (05-HM, Bio-Optica, Milano, Italy) for observation with a camera-coupled bright-field microscope.

    Protein Extraction:

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)
    Article Snippet: For detection of biotinylated proteins, streptavidin-HRP (#21126, Pierce) was utilized. .. For the assessment of cellular LRRC32 expression in the context of the signal peptide deletion constructs, an identical protocol as above was used except that cells were lysed using the M-PER Mammalian Protein Extraction Reagent (#78503, Pierce).

    Immunostaining:

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: Paragraph title: Tissue preparation and immunostaining. ... Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845).

    Lysis:

    Article Title: Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases
    Article Snippet: After precleared with streptavidin agarose (Invitrogen, Carlsbad, CA), the lysis was split equally into several parts and incubated with DMSO control or the probe with or without competitor for 30 min, followed by UV exposure at 365 nm for 60 min or not. .. Western Blot was performed as we reported previously using streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2 (Cell Signaling Technology, #4290, Boston, MA) antibodies.

    Plasmid Preparation:

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. .. Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used.

    Article Title: Loss of complex O-glycosylation impairs exocrine pancreatic function and induces MODY8-like diabetes in mice
    Article Snippet: Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. .. Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used.

    Software:

    Article Title: Mast cells are required for experimental oral allergen-induced diarrhea
    Article Snippet: After 30 minutes of incubation and one wash, streptavidin-HRP (1:20,000 dilution) (Immuno Pure Streptavidin Horseradish Peroxidase Conjugated II, 21126; Pierce Chemical Co.) was added. .. Plates were read using the Fluoroskan luminometer (Luminoskan, Ascent software, Thermo Electron Corporation, Franklin, Massachusetts, USA).

    Article Title: Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer
    Article Snippet: Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well. .. The plates were incubated for 8 min and the reaction was stopped with 100 μL of 2 M sulfuric acid before reading the optical density (OD) at 450 nm in a 96-well reader (Spectramax190, Molecular Devices, Sunnyvale, CA, USA) from Molecular Devices using SoftmaxPro software (Molecular Devices, Sunnyvale, CA).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Mast cells are required for experimental oral allergen-induced diarrhea
    Article Snippet: Paragraph title: ELISA measurements. ... After 30 minutes of incubation and one wash, streptavidin-HRP (1:20,000 dilution) (Immuno Pure Streptavidin Horseradish Peroxidase Conjugated II, 21126; Pierce Chemical Co.) was added.

    Article Title: Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer
    Article Snippet: Briefly, 100 μL of the mouse anti-human TF antibody, hTF1 (5 μg mL−1 in PBS), was coated onto 96-well ELISA plates (Nunc No. 439454) by overnight incubation at 4 °C. .. Streptavidin (Pierce No. 21126) (Pierce, Rockford, IL, USA) was diluted 1:10 000 and 100 μL was added to each well.

    Binding Assay:

    Article Title: Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination
    Article Snippet: Sections were permeabilized in 0.3% Triton X-100 and then blocked against nonspecific binding as described above. .. Sections were incubated with MOG primary antibody (1:1000, Abcam ab32760, RRID:AB_2145529) overnight followed by incubation in a biotinylated GαRb secondary antibody (1:500, Vector Laboratories BA-1000, RRID:AB_2313606) for 2 h, streptavidin (1 μg/ml, Pierce 21126) for an additional 2 h. Positive signal was detected using the NovaRed substrate kit (Vector Laboratories SK-4800, RRID:AB_2336845).

    Quantitation Assay:

    Article Title: Substrate Reduction Augments the Efficacy of Enzyme Therapy in a Mouse Model of Fabry Disease
    Article Snippet: Paragraph title: Quantitation of urine uromodulin ... Following incubation with streptavidin-HRP (Cat# 21127, Thermo Scientific, Rockland, IL) signal was detected using TMB HRP substrate (BioFX Laboratories, Inc., Owings Mills, MD).

    Immunoprecipitation:

    Article Title: Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases
    Article Snippet: Biological evaluation The pull down assay was carried out using the Immunoprecipitation Kit (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions with minor modifications. .. Western Blot was performed as we reported previously using streptavidin-HRP (Thermo Scientific, #21126, Waltham, MA), or EGFR (Cell Signaling Technology, #2232, Boston, MA), and HER2 (Cell Signaling Technology, #4290, Boston, MA) antibodies.

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)
    Article Snippet: Paragraph title: Biotinylation of cell surface proteins, Immunoprecipitation and Western Blotting ... For detection of biotinylated proteins, streptavidin-HRP (#21126, Pierce) was utilized.

    Staining:

    Article Title: Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury
    Article Snippet: MMP14 staining was detected with a rabbit monoclonal anti-MMP14 antibody (ab51074, Abcam) incubated overnight at 4 °C. .. Then, samples were incubated with the secondary antibody biotin-goat anti-rabbit IgG (H + L), followed by incubation with streptavidin–HRP (Cat #21126; Pierce).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher conjugated streptavidin
    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by <t>PE-streptavidin</t> and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P
    Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Streptavidin Conjugated Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated hrp - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher peroxidase conjugated streptavidin
    Assay principle. Interferon beta (IFN-β) 1a is non-specifically bound to the surface allowing the presentation of multiple epitopes. A “bridge” is formed by the subsequent addition of a positive serum sample or rabbit antihuman IFN-β and biotin-labeled IFN-β 1a. The latter is detected by HRP-conjugated <t>streptavidin.</t>
    Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    peroxidase conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    Image Search Results


    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by PE-streptavidin and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P

    Journal: Journal of Leukocyte Biology

    Article Title: CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells

    doi: 10.1189/jlb.2A0414-193RR

    Figure Lengend Snippet: Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by PE-streptavidin and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P

    Article Snippet: Reagents and antibodies Tissue-culture reagents, all secondary antibodies, and conjugated-streptavidin were purchased from Thermo Fisher Scientific (Paisley, Renfrewshire, United Kingdom).

    Techniques: Binding Assay, Labeling, Concentration Assay, Flow Cytometry, Cytometry, Whisker Assay, Derivative Assay

    Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Journal: Biochemistry

    Article Title: Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor

    doi: 10.1021/bi4014825

    Figure Lengend Snippet: Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Article Snippet: The resulting photoaffinity-labeled molecules were run on SDS-PAGE, transferred to PVDF, blocked with a 2.5% solution of bovine serum albumin (Fisher Scientific, Pittsburgh, PA), and incubated with streptavidin-conjugated HRP (ThermoFisher Scientific, Rockford, IL) at a 1:10 000 dilution before being washed with TBST and developed with SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific, Rockford, IL).

    Techniques: Labeling, Incubation

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Article Snippet: Samples were subsequently immunoprecipitated with an anti-GSK3β antibody and separated on SDS–PAGE followed by streptavidin conjugated to HRP (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing

    Assay principle. Interferon beta (IFN-β) 1a is non-specifically bound to the surface allowing the presentation of multiple epitopes. A “bridge” is formed by the subsequent addition of a positive serum sample or rabbit antihuman IFN-β and biotin-labeled IFN-β 1a. The latter is detected by HRP-conjugated streptavidin.

    Journal: Frontiers in Neurology

    Article Title: Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    doi: 10.3389/fneur.2017.00305

    Figure Lengend Snippet: Assay principle. Interferon beta (IFN-β) 1a is non-specifically bound to the surface allowing the presentation of multiple epitopes. A “bridge” is formed by the subsequent addition of a positive serum sample or rabbit antihuman IFN-β and biotin-labeled IFN-β 1a. The latter is detected by HRP-conjugated streptavidin.

    Article Snippet: After washing, 100 µL/well of horseradish peroxidase-conjugated streptavidin (Strep-HRP, 1 in 10,000, Thermo Fisher Scientific Pierce Technology, USA) is added.

    Techniques: Labeling