streptavidin hrp conjugate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin hrp conjugate
    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter"

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

    Journal: ACS nano

    doi: 10.1021/nn404945r

    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
    Figure Legend Snippet: GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Techniques Used: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Transmission Assay, Transmission Electron Microscopy, Labeling, Immunolabeling, Dot Blot, Isolation

    2) Product Images from "ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis"

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103089

    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).
    Figure Legend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Techniques Used: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

    Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.
    Figure Legend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo

    3) Product Images from "Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos"

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043434

    Hx-BP labels active MMPs in vitro . 135 ng of human recombinant MMP-2 (hrMMP-2) was incubated with 0.5 nmol of biotinylated HxBP, with or without activation by APMA and with or without UV crosslinking, then resolved by SDS-PAGE and blotted to PVDF membrane. Streptavidin-HRP detection of biotinylated hrMMP-2 is dependent on both the activation status of the protease, and on exposure to UV light (h v ).
    Figure Legend Snippet: Hx-BP labels active MMPs in vitro . 135 ng of human recombinant MMP-2 (hrMMP-2) was incubated with 0.5 nmol of biotinylated HxBP, with or without activation by APMA and with or without UV crosslinking, then resolved by SDS-PAGE and blotted to PVDF membrane. Streptavidin-HRP detection of biotinylated hrMMP-2 is dependent on both the activation status of the protease, and on exposure to UV light (h v ).

    Techniques Used: In Vitro, Recombinant, Incubation, Activation Assay, SDS Page

    HxBP labels its target proteins in vivo . Live Xenopus tadpoles were injected with HxBP-alkyne, allowed to recover, exposed to UV light, and then sacrificed and protein extracts of the tails were subjected to click chemistry to biotinylate HxBP-tagged proteins. In panel A, biotinylated proteins were affinity purified by avidin chromatography, resolved by SDS-PAGE and detected by silver staining. A single protein with a mobility consistent with xMMP2 is detectable in the lane containing the HxBP-labeled proteome of the larvae that were induced to express xMMP2 by treatment with T3, and then biotinylated by click chemistry. In panel B, tail homogenates were split and run in parallel on a gelatin zymography gel (left) and for blotting to PVDF (right). Blots were probed with streptavidin-HRP, and revealed a single labeled protein at the same mobility as the gelatinolytic xMMP2 band on the zymograph.
    Figure Legend Snippet: HxBP labels its target proteins in vivo . Live Xenopus tadpoles were injected with HxBP-alkyne, allowed to recover, exposed to UV light, and then sacrificed and protein extracts of the tails were subjected to click chemistry to biotinylate HxBP-tagged proteins. In panel A, biotinylated proteins were affinity purified by avidin chromatography, resolved by SDS-PAGE and detected by silver staining. A single protein with a mobility consistent with xMMP2 is detectable in the lane containing the HxBP-labeled proteome of the larvae that were induced to express xMMP2 by treatment with T3, and then biotinylated by click chemistry. In panel B, tail homogenates were split and run in parallel on a gelatin zymography gel (left) and for blotting to PVDF (right). Blots were probed with streptavidin-HRP, and revealed a single labeled protein at the same mobility as the gelatinolytic xMMP2 band on the zymograph.

    Techniques Used: In Vivo, Injection, Affinity Purification, Avidin-Biotin Assay, Chromatography, SDS Page, Silver Staining, Labeling, Zymography

    4) Product Images from "Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor"

    Article Title: Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3019

    Minor amounts of intermolecular proteolysis are observable in vitro . Western blot of C‐terminally biotinylated ProCat(S386A), which was incubated alone, and in the presence of ProCat(WT) Processed , ProCat(Q152H) zymogen , ProCat(Q152N) zymogen , and ProCat(Q152N) Processed , denoted by “WT P ”, “Q152H Z ”, “Q152N Z ”, and “Q152N P ”, respectively. The western was visualized using a streptavidin‐HRP conjugate.
    Figure Legend Snippet: Minor amounts of intermolecular proteolysis are observable in vitro . Western blot of C‐terminally biotinylated ProCat(S386A), which was incubated alone, and in the presence of ProCat(WT) Processed , ProCat(Q152H) zymogen , ProCat(Q152N) zymogen , and ProCat(Q152N) Processed , denoted by “WT P ”, “Q152H Z ”, “Q152N Z ”, and “Q152N P ”, respectively. The western was visualized using a streptavidin‐HRP conjugate.

    Techniques Used: In Vitro, Western Blot, Incubation

    5) Product Images from "Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling"

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling

    Journal: Chemistry & biology

    doi: 10.1016/j.chembiol.2005.07.006

    Detection of TfR1-PCP Using Biotin Labeling TRVb/TfR1-PCP or TRVb cells were incubated with or without biotin-CoA (1) in the presence and absence of Sfp. Cell lysates were prepared for electrophoresis under both nonreducing (A) and reducing (B) conditions. After transfer to a PVDF membrane, labeling with biotin was probed using streptavidin-HRP.
    Figure Legend Snippet: Detection of TfR1-PCP Using Biotin Labeling TRVb/TfR1-PCP or TRVb cells were incubated with or without biotin-CoA (1) in the presence and absence of Sfp. Cell lysates were prepared for electrophoresis under both nonreducing (A) and reducing (B) conditions. After transfer to a PVDF membrane, labeling with biotin was probed using streptavidin-HRP.

    Techniques Used: Labeling, Incubation, Electrophoresis

    6) Product Images from "Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation"

    Article Title: Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-014-5715-6

    The purified Δ148aa-hACC2 possesses posttranslational biotinylation confirmed by Western blot analysis using an anti-biotin antibody ( A ) and streptavidin HRP conjugate ( B ). MW , molecular weight markers; Lanes 1 and 4 , protein extracts after infection; Lanes 2 and 5 , flow through during FLAG-tag purification; Lanes 3 and 6 , purified and concentrated Δ148aa-hACC2. An anti-biotin antibody and a streptavidin HRP conjugate were used as primary antibodies. A rabbit anti-goat IgG-HRP and an anti-mouse IgG-HRP were used as secondary antibodies
    Figure Legend Snippet: The purified Δ148aa-hACC2 possesses posttranslational biotinylation confirmed by Western blot analysis using an anti-biotin antibody ( A ) and streptavidin HRP conjugate ( B ). MW , molecular weight markers; Lanes 1 and 4 , protein extracts after infection; Lanes 2 and 5 , flow through during FLAG-tag purification; Lanes 3 and 6 , purified and concentrated Δ148aa-hACC2. An anti-biotin antibody and a streptavidin HRP conjugate were used as primary antibodies. A rabbit anti-goat IgG-HRP and an anti-mouse IgG-HRP were used as secondary antibodies

    Techniques Used: Purification, Western Blot, Molecular Weight, Infection, Flow Cytometry, FLAG-tag

    7) Product Images from "Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics"

    Article Title: Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110316

    Affinity purification of biotinylated cell surface sialoglycoproteins. U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac 4 ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6 7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6 7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
    Figure Legend Snippet: Affinity purification of biotinylated cell surface sialoglycoproteins. U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac 4 ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6 7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6 7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).

    Techniques Used: Affinity Purification, Metabolic Labelling, SDS Page, Flow Cytometry, Western Blot, Immunodetection, Staining, Expressing

    8) Product Images from "Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation "

    Article Title: Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation

    Journal: The Journal of Cell Biology

    doi:

    Schematic representation of human E-cadherin gene constructs ( a ) and sedimentation/coimmunoprecipitation analysis showing E-cadherin dimerization ( b and c ). ( a ) Five extracellular cadherin-like repeats (numbered I–V), the intracellular p120-binding site ( P ), the catenin-binding domain ( C ), and myc ( fused solid triangles ) or flag ( solid square ) epitopes are indicated. Deletions are depicted by brackets . Leader propeptide ( Leader ) is shown by the dotted line . Numbers show positions of the corresponding amino acids. ( b ) Fractions of the total lysate of the A-431 cells producing Ec1M were separated in a sucrose gradient. The fractions were then coimmunoprecipitated with anti-myc mAb and analyzed by immunoblotting with anti-myc and anti–E-cadherin mAbs for the presence of Ec1M ( Ec1M ) and endogenous E-cadherin ( Ec ), respectively. Sedimentation of the Ec1M–E-cadherin complex is the same as the plakoglobin–β-catenin complex (refer to Fig. 1 a ). ( c ) Surface proteins of Ec1M-expressing cells were biotinylated and then the lysate was separated by sucrose gradient and fractions were immunoprecipitated by anti–E-cadherin mAb. Only the 13 S (fraction 6 ) and 9 S (fraction 8 ) fractions are shown. Immunoprecipitates were analyzed by immunoblotting using streptavidin-HRP conjugate ( Str., HRP ), anti-myc ( myc ), anti– E-cadherin, and anti–β-catenin antibodies. Note that anti–E-cadherin antibody coimmunoprecipitates biotinylated Ec1M only in fraction 6 . This coimmunoprecipitation is stable after dissociation of the labeled cells into single-cell suspension (lane 6′ ). The absence of catenin biotinylation in E-cadherin–catenin complexes indicates specific incorporation of biotin only into surface proteins. Positions of endogenous E-cadherin ( Ec ) and Ec1M are indicated by arrows . Molecular weight markers are shown in kD.
    Figure Legend Snippet: Schematic representation of human E-cadherin gene constructs ( a ) and sedimentation/coimmunoprecipitation analysis showing E-cadherin dimerization ( b and c ). ( a ) Five extracellular cadherin-like repeats (numbered I–V), the intracellular p120-binding site ( P ), the catenin-binding domain ( C ), and myc ( fused solid triangles ) or flag ( solid square ) epitopes are indicated. Deletions are depicted by brackets . Leader propeptide ( Leader ) is shown by the dotted line . Numbers show positions of the corresponding amino acids. ( b ) Fractions of the total lysate of the A-431 cells producing Ec1M were separated in a sucrose gradient. The fractions were then coimmunoprecipitated with anti-myc mAb and analyzed by immunoblotting with anti-myc and anti–E-cadherin mAbs for the presence of Ec1M ( Ec1M ) and endogenous E-cadherin ( Ec ), respectively. Sedimentation of the Ec1M–E-cadherin complex is the same as the plakoglobin–β-catenin complex (refer to Fig. 1 a ). ( c ) Surface proteins of Ec1M-expressing cells were biotinylated and then the lysate was separated by sucrose gradient and fractions were immunoprecipitated by anti–E-cadherin mAb. Only the 13 S (fraction 6 ) and 9 S (fraction 8 ) fractions are shown. Immunoprecipitates were analyzed by immunoblotting using streptavidin-HRP conjugate ( Str., HRP ), anti-myc ( myc ), anti– E-cadherin, and anti–β-catenin antibodies. Note that anti–E-cadherin antibody coimmunoprecipitates biotinylated Ec1M only in fraction 6 . This coimmunoprecipitation is stable after dissociation of the labeled cells into single-cell suspension (lane 6′ ). The absence of catenin biotinylation in E-cadherin–catenin complexes indicates specific incorporation of biotin only into surface proteins. Positions of endogenous E-cadherin ( Ec ) and Ec1M are indicated by arrows . Molecular weight markers are shown in kD.

    Techniques Used: Construct, Sedimentation, Binding Assay, Expressing, Immunoprecipitation, Labeling, Molecular Weight

    9) Product Images from "Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy"

    Article Title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy

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

    doi: 10.1073/pnas.1204523109

    EF2 refractory to ADP ribosylation renders resistant cells nonresponsive to HA22. ( A ) CD22 expression. Cells were incubated on ice with RFB4 (mouse anti-CD22 antibody) or control mouse IgG1, followed by goat anti-mouse antibody-phycoerythrin (PE) and analyzed by FACS. MFI, geometric mean of fluorescence intensity. ( B ) Time course of immunotoxin internalization. HAL-01 and HAL-01-R cells were incubated with HA22-Alexa 647 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 does not inhibit protein synthesis in resistant cell line HAL-01-R. 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 can ADP ribosylate EF2 in parental cells but not in resistant 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 blot with streptavidin HRP conjugate to detect biotin-ADP ribose-EF2. EF2 and actin are shown as loading controls.
    Figure Legend Snippet: EF2 refractory to ADP ribosylation renders resistant cells nonresponsive to HA22. ( A ) CD22 expression. Cells were incubated on ice with RFB4 (mouse anti-CD22 antibody) or control mouse IgG1, followed by goat anti-mouse antibody-phycoerythrin (PE) and analyzed by FACS. MFI, geometric mean of fluorescence intensity. ( B ) Time course of immunotoxin internalization. HAL-01 and HAL-01-R cells were incubated with HA22-Alexa 647 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 does not inhibit protein synthesis in resistant cell line HAL-01-R. 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 can ADP ribosylate EF2 in parental cells but not in resistant 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 blot with streptavidin HRP conjugate to detect biotin-ADP ribose-EF2. EF2 and actin are shown as loading controls.

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

    10) Product Images from "Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿"

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00911-09

    2D gel and blot of biotin-labeled R. parkeri surface proteins. The biotinylated proteins resolved by 2D PAGE were stained with SYPRO Ruby protein gel stain (A) or transferred to a PVDF membrane and detected using streptavidin-HRP conjugate (B). The numbers
    Figure Legend Snippet: 2D gel and blot of biotin-labeled R. parkeri surface proteins. The biotinylated proteins resolved by 2D PAGE were stained with SYPRO Ruby protein gel stain (A) or transferred to a PVDF membrane and detected using streptavidin-HRP conjugate (B). The numbers

    Techniques Used: Two-Dimensional Gel Electrophoresis, Labeling, Polyacrylamide Gel Electrophoresis, Staining

    11) Product Images from "ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis"

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103089

    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).
    Figure Legend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Techniques Used: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

    Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.
    Figure Legend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo

    12) Product Images from "Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes"

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-11-20

    Direct sandwich ELISA format for the detection of the phosphorylated DYRK substrate peptide tau 207-219 . A , Scheme illustrating the principle of the assay. Bio, biotin; TMB, tetramethylbenzidine; HRP, horseradish peroxidase (coupled to streptavidin). B and C , Titration of phosphorylated and unphosphorylated tau 207-219 . The wells were coated with 100 ng anti tau(pT212) and loaded with dilution series of either phosphorylated or unphosphorylated tau 207-219 . The background signal from wells loaded only with the buffer was subtracted from all values. A representative experiment of three is shown. Panel C presents the same data as in panel B with a linear x-axis to visualize the linear range of the ELISA. The inset shows an enlargement of lower range. D , Titration of phosphorylated and non-phosphorylated tau 207-219 on streptavidin-coated wells. Detection was performed with primary anti-tau(pT212) antibody and secondary goat anti-rabbit antibody coupled to HRP. The graph is representative of two experiments. E , Detection of phosphorylated tau 207-219 in the presence of excess unphosphorylated peptide. Different amounts of phosphorylated tau 207-219 (0.01 pmol, 0.1 pmol, 1 pmol) were mixed with a dilution series of unphosphorylated tau 207-219 (12.5 - 100 pmol). Signals obtained in wells loaded only with the same amount of the unphosphorylated peptide were subtracted from the read-out of the mixtures. The graph is representative of two experiments. In B-E, error bars indicate the difference between duplicate wells.
    Figure Legend Snippet: Direct sandwich ELISA format for the detection of the phosphorylated DYRK substrate peptide tau 207-219 . A , Scheme illustrating the principle of the assay. Bio, biotin; TMB, tetramethylbenzidine; HRP, horseradish peroxidase (coupled to streptavidin). B and C , Titration of phosphorylated and unphosphorylated tau 207-219 . The wells were coated with 100 ng anti tau(pT212) and loaded with dilution series of either phosphorylated or unphosphorylated tau 207-219 . The background signal from wells loaded only with the buffer was subtracted from all values. A representative experiment of three is shown. Panel C presents the same data as in panel B with a linear x-axis to visualize the linear range of the ELISA. The inset shows an enlargement of lower range. D , Titration of phosphorylated and non-phosphorylated tau 207-219 on streptavidin-coated wells. Detection was performed with primary anti-tau(pT212) antibody and secondary goat anti-rabbit antibody coupled to HRP. The graph is representative of two experiments. E , Detection of phosphorylated tau 207-219 in the presence of excess unphosphorylated peptide. Different amounts of phosphorylated tau 207-219 (0.01 pmol, 0.1 pmol, 1 pmol) were mixed with a dilution series of unphosphorylated tau 207-219 (12.5 - 100 pmol). Signals obtained in wells loaded only with the same amount of the unphosphorylated peptide were subtracted from the read-out of the mixtures. The graph is representative of two experiments. In B-E, error bars indicate the difference between duplicate wells.

    Techniques Used: Sandwich ELISA, Titration, Enzyme-linked Immunosorbent Assay

    13) Product Images from "TDP-43 regulates site-specific 2′-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs"

    Article Title: TDP-43 regulates site-specific 2′-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz086

    TDP-43 binds C/D scaRNAs that have a UG-rich motif. ( A ) Isolation of RNAs bound to DAP-TDP-43 by two-step affinity purification (pull-down, PD; His-tag and FLAG-tag) from the cell extract of DAP-TDP-43-expressing T-REx 293 cells after a 24-h treatment with doxycycline. DAP-TDP-43 was detected by western blotting using horseradish peroxidase-conjugated streptavidin (streptavidin-HRP; Biotin). scaRNA2 and scaRNA28 were identified by in-gel RNase T1 digestion–LC-MS analysis. Input, total RNA (1 μg/cell line). ( B ) Oligonucleotides identified by LC-MS analysis of the in-gel RNase T1-digested RNA bands corresponding to scaRNA2 and scaRNA28 in Figure 1A are underlined in the sequence of scaRNA2 and scaRNA28. Red lines indicate oligonucleotides identified by Ariadne search, and green lines are by manual inspection. ( C ) Schematic structures of C/D scaRNAs (scaRNA2, 7, 9, 28 and 17). UG-rich motif and the guide domain for post-transcriptional modification are shown in each scaRNA. DNA probes used for northern blot analysis are indicated as black lines under each scaRNA. ( D ) DAP-TDP-43−binding RNAs immunoprecipitated using FLAG antibody (IP: FLAG) were detected by northern blot analysis with DNA probes hybridizing to the RNAs indicated on the right. The parental T-REx 293 cells and DAP-LYAR-expressing T-REx 293 cells were used as controls. DAP-TDP-43 and DAP-LYAR were detected by western blot analysis using streptavidin-HRP (Biotin). Input, total RNA (4 μg/cell line). ( E ) scaRNAs were isolated by the FLAG immunoprecipitation from DAP-TDP-43-expressing T-REx 293 cells with (+) or without (−) UV irradiation, separated by SDS-PGAE (left) and analyzed by northern blot analysis using DNA probes hybridizing to the RNAs indicated on the right (right). Half bracket indicates the position of the excised membrane. RNA-unbound DAP-TDP-43 detected by streptavidin-HRP is indicated by an asterisk. ( F ) Electrophoresis mobility shift assay using each of the biotin-labeled scaRNAs was done with (+) or without (−) recombinant TDP-43 (TF-TDP-43-FL), TF-FL and (UG) 6 RNA oligonucleotide. ( G ) Schematic structures of DAP-TDP-43 deletion mutants (top). T-REx 293 cells or T-REx 293 cells expressing DAP-TDP-43 (WT) or its deletion mutants (Δ315, ΔGR, ΔRRM2 and ΔRRM1) were treated for 24 h with 100 ng/ml of doxycycline (dox), and the expression levels of DAP-TDP-43, its deletion mutants or endogenous TDP-43 were detected by western blot analysis with anti-TDP-43 antibodies (mouse monoclonal antibody, mAb; rabbit polyclonal antibody, rAb). GAPDH was used as a loading control. Open arrowhead shows endogenous TDP-43. ( H ) RNAs immunoprecipitated with DAP-TDP-43 (WT) and its deletion mutants (Δ315, ΔGR, ΔRRM1, ΔRRM2) using FLAG antibody (IP: FLAG) were detected by northern blot analysis with DNA probes hybridizing to the RNAs indicated on the right. The parental T-REx 293 cells were used as a control. DAP-TDP-43 and its deletion mutants were detected by western blot analysis using streptavidin-HRP (Biotin). Input, total RNA (4 μg/cell line). ( I ) T-REx 293 cells expressing TDP-43 (WT) or domain mutants (Δ315, ΔGR, ΔRRM2 and ΔRRM1) were treated with (+) or without (−) doxycycline for 24 h. Total RNAs were analyzed by northern blotting using DNA probes hybridizing to the RNAs indicated to the right. Ratios of staining band intensities (+dox/−dox) are shown by bar graph for the cells expressing TDP-43 or each of the domain mutants. Mean ± SEM, n = 3; * P
    Figure Legend Snippet: TDP-43 binds C/D scaRNAs that have a UG-rich motif. ( A ) Isolation of RNAs bound to DAP-TDP-43 by two-step affinity purification (pull-down, PD; His-tag and FLAG-tag) from the cell extract of DAP-TDP-43-expressing T-REx 293 cells after a 24-h treatment with doxycycline. DAP-TDP-43 was detected by western blotting using horseradish peroxidase-conjugated streptavidin (streptavidin-HRP; Biotin). scaRNA2 and scaRNA28 were identified by in-gel RNase T1 digestion–LC-MS analysis. Input, total RNA (1 μg/cell line). ( B ) Oligonucleotides identified by LC-MS analysis of the in-gel RNase T1-digested RNA bands corresponding to scaRNA2 and scaRNA28 in Figure 1A are underlined in the sequence of scaRNA2 and scaRNA28. Red lines indicate oligonucleotides identified by Ariadne search, and green lines are by manual inspection. ( C ) Schematic structures of C/D scaRNAs (scaRNA2, 7, 9, 28 and 17). UG-rich motif and the guide domain for post-transcriptional modification are shown in each scaRNA. DNA probes used for northern blot analysis are indicated as black lines under each scaRNA. ( D ) DAP-TDP-43−binding RNAs immunoprecipitated using FLAG antibody (IP: FLAG) were detected by northern blot analysis with DNA probes hybridizing to the RNAs indicated on the right. The parental T-REx 293 cells and DAP-LYAR-expressing T-REx 293 cells were used as controls. DAP-TDP-43 and DAP-LYAR were detected by western blot analysis using streptavidin-HRP (Biotin). Input, total RNA (4 μg/cell line). ( E ) scaRNAs were isolated by the FLAG immunoprecipitation from DAP-TDP-43-expressing T-REx 293 cells with (+) or without (−) UV irradiation, separated by SDS-PGAE (left) and analyzed by northern blot analysis using DNA probes hybridizing to the RNAs indicated on the right (right). Half bracket indicates the position of the excised membrane. RNA-unbound DAP-TDP-43 detected by streptavidin-HRP is indicated by an asterisk. ( F ) Electrophoresis mobility shift assay using each of the biotin-labeled scaRNAs was done with (+) or without (−) recombinant TDP-43 (TF-TDP-43-FL), TF-FL and (UG) 6 RNA oligonucleotide. ( G ) Schematic structures of DAP-TDP-43 deletion mutants (top). T-REx 293 cells or T-REx 293 cells expressing DAP-TDP-43 (WT) or its deletion mutants (Δ315, ΔGR, ΔRRM2 and ΔRRM1) were treated for 24 h with 100 ng/ml of doxycycline (dox), and the expression levels of DAP-TDP-43, its deletion mutants or endogenous TDP-43 were detected by western blot analysis with anti-TDP-43 antibodies (mouse monoclonal antibody, mAb; rabbit polyclonal antibody, rAb). GAPDH was used as a loading control. Open arrowhead shows endogenous TDP-43. ( H ) RNAs immunoprecipitated with DAP-TDP-43 (WT) and its deletion mutants (Δ315, ΔGR, ΔRRM1, ΔRRM2) using FLAG antibody (IP: FLAG) were detected by northern blot analysis with DNA probes hybridizing to the RNAs indicated on the right. The parental T-REx 293 cells were used as a control. DAP-TDP-43 and its deletion mutants were detected by western blot analysis using streptavidin-HRP (Biotin). Input, total RNA (4 μg/cell line). ( I ) T-REx 293 cells expressing TDP-43 (WT) or domain mutants (Δ315, ΔGR, ΔRRM2 and ΔRRM1) were treated with (+) or without (−) doxycycline for 24 h. Total RNAs were analyzed by northern blotting using DNA probes hybridizing to the RNAs indicated to the right. Ratios of staining band intensities (+dox/−dox) are shown by bar graph for the cells expressing TDP-43 or each of the domain mutants. Mean ± SEM, n = 3; * P

    Techniques Used: Isolation, Affinity Purification, FLAG-tag, Expressing, Western Blot, Liquid Chromatography with Mass Spectroscopy, Sequencing, Modification, Northern Blot, Binding Assay, Immunoprecipitation, Irradiation, Electrophoresis, Mobility Shift, Labeling, Recombinant, Staining

    WDR79 is not involved in CB localization of UG-rich motif-bearing C/D scaRNAs. ( A ) Isolation of WDR79−TDP-43 complex by two-step pull-down (PD) method. DAP-TDP-43 complexes were first pulled down (1st-PD) with anti-FLAG from DAP-TDP-43-expressing T-REx 293 cells (induced with doxycycline for 24 h) and then pulled down (2nd-PD) with anti-WDR79 (protocol is shown at left). Protein components were detected by western blot analysis (IB) with anti-WDR79 or streptavidin-HRP (biotin). RNAs were detected by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input consisted of 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. ( B ) HEF-WDR79-expressing T-REx 293 cells were treated with ncRNA (nc) or TDP-43 siRNA (si) for 72 h and further treated with Dox for 24 h. RNAs and proteins associated with HEF-WDR79 were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Protein components were detected by western blot analysis (IB), and RNAs were by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input, 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. The graph shows the band intensities of the scaRNAs immunoprecipitated from siRNA-treated cells relative to those from ncRNA-treated cells. The values are normalized with those of the immunoprecipitated (IPed) HEF-WDR79. Mean ± SEM, n = 3; * P
    Figure Legend Snippet: WDR79 is not involved in CB localization of UG-rich motif-bearing C/D scaRNAs. ( A ) Isolation of WDR79−TDP-43 complex by two-step pull-down (PD) method. DAP-TDP-43 complexes were first pulled down (1st-PD) with anti-FLAG from DAP-TDP-43-expressing T-REx 293 cells (induced with doxycycline for 24 h) and then pulled down (2nd-PD) with anti-WDR79 (protocol is shown at left). Protein components were detected by western blot analysis (IB) with anti-WDR79 or streptavidin-HRP (biotin). RNAs were detected by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input consisted of 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. ( B ) HEF-WDR79-expressing T-REx 293 cells were treated with ncRNA (nc) or TDP-43 siRNA (si) for 72 h and further treated with Dox for 24 h. RNAs and proteins associated with HEF-WDR79 were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Protein components were detected by western blot analysis (IB), and RNAs were by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input, 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. The graph shows the band intensities of the scaRNAs immunoprecipitated from siRNA-treated cells relative to those from ncRNA-treated cells. The values are normalized with those of the immunoprecipitated (IPed) HEF-WDR79. Mean ± SEM, n = 3; * P

    Techniques Used: Isolation, Expressing, Western Blot, Northern Blot, Immunoprecipitation

    14) Product Images from "ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis"

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103089

    Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.
    Figure Legend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo

    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).
    Figure Legend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Techniques Used: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

    15) Product Images from "Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics"

    Article Title: Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110316

    Affinity purification of biotinylated cell surface sialoglycoproteins. U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac 4 ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6 7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6 7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
    Figure Legend Snippet: Affinity purification of biotinylated cell surface sialoglycoproteins. U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac 4 ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6 7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6 7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).

    Techniques Used: Affinity Purification, Metabolic Labelling, SDS Page, Flow Cytometry, Western Blot, Immunodetection, Staining, Expressing

    16) Product Images from "ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis"

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103089

    Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.
    Figure Legend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo

    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).
    Figure Legend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Techniques Used: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

    17) Product Images from "Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line"

    Article Title: Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122462

    Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R. A : Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [ 3 H]leucine incorporation. B : Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C : EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D : EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.
    Figure Legend Snippet: Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R. A : Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [ 3 H]leucine incorporation. B : Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C : EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D : EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.

    Techniques Used: Inhibition, Incubation, Expressing, Quantitative RT-PCR, Functional Assay, SDS Page, Western Blot

    18) Product Images from "Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation"

    Article Title: Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-014-5715-6

    The purified Δ148aa-hACC2 possesses posttranslational biotinylation confirmed by Western blot analysis using an anti-biotin antibody ( A ) and streptavidin HRP conjugate ( B ). MW , molecular weight markers; Lanes 1 and 4 , protein extracts after infection; Lanes 2 and 5 , flow through during FLAG-tag purification; Lanes 3 and 6 , purified and concentrated Δ148aa-hACC2. An anti-biotin antibody and a streptavidin HRP conjugate were used as primary antibodies. A rabbit anti-goat IgG-HRP and an anti-mouse IgG-HRP were used as secondary antibodies
    Figure Legend Snippet: The purified Δ148aa-hACC2 possesses posttranslational biotinylation confirmed by Western blot analysis using an anti-biotin antibody ( A ) and streptavidin HRP conjugate ( B ). MW , molecular weight markers; Lanes 1 and 4 , protein extracts after infection; Lanes 2 and 5 , flow through during FLAG-tag purification; Lanes 3 and 6 , purified and concentrated Δ148aa-hACC2. An anti-biotin antibody and a streptavidin HRP conjugate were used as primary antibodies. A rabbit anti-goat IgG-HRP and an anti-mouse IgG-HRP were used as secondary antibodies

    Techniques Used: Purification, Western Blot, Molecular Weight, Infection, Flow Cytometry, FLAG-tag

    19) Product Images from "Enzymatic Characterization of a Prokaryotic Urea Carboxylase"

    Article Title: Enzymatic Characterization of a Prokaryotic Urea Carboxylase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.9.2532-2539.2004

    Presence of multiple biotinylated proteins in O. sagaranesis . Biotinylated proteins in the cell extracts of O. sagaranensis were partially purified with TetraLink tetrameric avidin resin as described in Materials and Methods. The partially purified extract was subjected to SDS-PAGE and detected with streptavidin-HRP conjugate. The arrowheads indicate the positions of three biotinylated proteins with molecular masses of approximately 130, 120, and 70 kDa.
    Figure Legend Snippet: Presence of multiple biotinylated proteins in O. sagaranesis . Biotinylated proteins in the cell extracts of O. sagaranensis were partially purified with TetraLink tetrameric avidin resin as described in Materials and Methods. The partially purified extract was subjected to SDS-PAGE and detected with streptavidin-HRP conjugate. The arrowheads indicate the positions of three biotinylated proteins with molecular masses of approximately 130, 120, and 70 kDa.

    Techniques Used: Purification, Avidin-Biotin Assay, SDS Page

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    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis
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    Sandwich ELISA:

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes
    Article Snippet: Paragraph title: Direct Sandwich ELISA ... Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed.

    Incubation:

    Article Title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy
    Article Snippet: Cells were lysed in 0.3 mL radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors, and 0.01 mL of cell lysate (30 μg) was incubated with 100 ng of HA22 in ADP ribosylation buffer [20 mM Tris·HCl (pH 7.4), 1 mM EDTA, and 50 mM DTT] with 5 mM 6-Biotin-17-NAD (Trevigen) for 60 min at 25 °C. .. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate (Invitrogen) to detect biotin-ADP ribose-EF2.

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C. .. The blot was then washed three times for 15 minutes in PBSTw to remove unbound streptavidin-HRP and detection of biotinylated proteins was performed using an enhanced chemiluminescence (ECL) kit (Pierce) according to the manufacturer’s directions.

    Article Title: Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor
    Article Snippet: .. The bands were transferred to a nitrocellulose membrane using an iBlot dry blotting system (Life Technologies) and were incubated with a streptavidin‐HRP conjugate (Life Technologies). .. The bands were visualized using SuperSignal west pico chemiluminescent substrate (Thermo Scientific).

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: .. The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr. ..

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes
    Article Snippet: .. Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed. .. Other detection reagents (HRP-linked anti-biotin antibody from CST; strepavidin-HRP from R & D systems) were less sensitive, and higher concentration of the streptavidin-HRP conjugate resulted in higher background in empty wells.

    Article Title: Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation
    Article Snippet: Each plate was incubated at 4°C with 7 ml of 0.5 mg/ml of sulfo-NHS-LC-biotin ( Pierce Chemical Co. , Rockford, IL) in PBS-C for 1 h. The reaction was quenched by washing the cells with 1 M Tris/100 mM glycine buffer, pH 7.5. .. Biotinylated proteins were visualized with streptavidin-HRP conjugate ( Pierce Chemical Co. ) in conjunction with enhanced chemiluminescence ( Boehringer Mannheim , Indianapolis, IN).

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter
    Article Snippet: .. Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ). .. Isolated EV-Gluc and EV-GlucB were diluted 1,000 fold with double-0.22 μm-membrane-filtered PBS and subjected to LM10 nanoparticle analyzer (NanoSight, Duxbury, MA) to determine size distribution using NTA software version 2.2.

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿
    Article Snippet: .. Rickettsial surface proteins were detected by incubation of the membrane with streptavidin-HRP conjugate (Invitrogen) at a dilution of 1:6,000 for 1 h at room temperature. ..

    Modification:

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes
    Article Snippet: After blocking for 1 hour with 200 μl/well 2% (w/v) bovine serum albumin (fraction V, modified Cohn, Calbiochem) in phosphate buffered saline (PBS) (140 mM NaCl, 3 mM KCl, 8 mM Na2 HPO4 , 1.8 mM KH2 PO4 , pH 7.4), the antigen was added in a volume of 100 μL/well and the plates were incubated for 2 h at room temperature. .. Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed.

    Western Blot:

    Article Title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy
    Article Snippet: .. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate (Invitrogen) to detect biotin-ADP ribose-EF2. .. Cells were collected, washed with cold Dulbecco's phosphate buffered saline (DPBS) twice, and solubilized in lysis buffer [25 nM Tris·HCl (pH 7.5), 150 nM NaCl, 1.0% Nonidet P-40 (vol/vol), and 0.5% sodium deoxycholate (wt/vol)) with protease inhibitor mixture (Sigma).

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: Paragraph title: Biotin Labeling and Western Blotting ... The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr.

    Article Title: Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics
    Article Snippet: .. Captured proteins were eluted from the beads and subjected to SDS-PAGE and Western blot analysis using a streptavidin-HRP conjugate. ..

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿
    Article Snippet: Paragraph title: Western blot analysis. ... Rickettsial surface proteins were detected by incubation of the membrane with streptavidin-HRP conjugate (Invitrogen) at a dilution of 1:6,000 for 1 h at room temperature.

    Electrophoresis:

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: After electrophoresis, the protein bands were electroblotted onto a piece of PVDF membrane (Biorad). .. The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr.

    other:

    Article Title: Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation
    Article Snippet: To further validate biotinylation of the Δ148aa-hACC2, streptavidin HRP conjugate was employed to detect the biotin group as streptavidin is known to interact with biotin with very high affinity.

    Binding Assay:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C. .. The blot was then washed three times for 15 minutes in PBSTw to remove unbound streptavidin-HRP and detection of biotinylated proteins was performed using an enhanced chemiluminescence (ECL) kit (Pierce) according to the manufacturer’s directions.

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr. .. Streptavidin binding was detected using an ECL luminescence detection kit (Amersham Pharmacia, Piscataway, NJ).

    Labeling:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: Paragraph title: In vitro Labeling of Human Recombinant MMP-2 ... The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: Paragraph title: Biotin Labeling and Western Blotting ... The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr.

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter
    Article Snippet: .. Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ). .. Isolated EV-Gluc and EV-GlucB were diluted 1,000 fold with double-0.22 μm-membrane-filtered PBS and subjected to LM10 nanoparticle analyzer (NanoSight, Duxbury, MA) to determine size distribution using NTA software version 2.2.

    Purification:

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis
    Article Snippet: .. The samples of bacterial lysates in PBSTB or purified proteins were applied as a series of different dilutions in PBSTB and bound biotinylated ARS proteins were detected using streptavidin-HRP conjugate diluted in the same buffer 1:10,000 (Pierce, Rockford, IL, USA). .. Results were visualized by enzymatic reaction of HRP with OPD substrate (Sigma-Aldrich, St. Luis, MO, USA) in citrate buffer (3.31% sodium citrate tribasic dihydrate, phosphoric acid until pH = 5.0), reaction was stopped by 2 M sulfuric acid and absorbance at 492 nm was measured.

    Dot Blot:

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter
    Article Snippet: Paragraph title: Dot blot detection of biotinylated EVs ... Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿
    Article Snippet: Unlabeled or biotin-labeled R. parkeri proteins separated by 2D PAGE were electroblotted onto Immun-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad) by using an XCell II blot module (Invitrogen) according to the manufacturer's protocol. .. Rickettsial surface proteins were detected by incubation of the membrane with streptavidin-HRP conjugate (Invitrogen) at a dilution of 1:6,000 for 1 h at room temperature.

    Lysis:

    Article Title: Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation
    Article Snippet: Then cells were lysed in lysis buffer and analyzed by sucrose gradient centrifugation and coimmunoprecipitation assays as described above. .. Biotinylated proteins were visualized with streptavidin-HRP conjugate ( Pierce Chemical Co. ) in conjunction with enhanced chemiluminescence ( Boehringer Mannheim , Indianapolis, IN).

    SDS Page:

    Article Title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy
    Article Snippet: .. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate (Invitrogen) to detect biotin-ADP ribose-EF2. .. Cells were collected, washed with cold Dulbecco's phosphate buffered saline (DPBS) twice, and solubilized in lysis buffer [25 nM Tris·HCl (pH 7.5), 150 nM NaCl, 1.0% Nonidet P-40 (vol/vol), and 0.5% sodium deoxycholate (wt/vol)) with protease inhibitor mixture (Sigma).

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: The samples were mixed 1∶1 in reducing SDS-PAGE sample buffer (225 mM Tris pH 6.8, 50% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.05% bromophenol blue). .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Article Title: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling
    Article Snippet: To detect biotin labeling, cell lysates containing 2 μg protein were loaded on a 4%–15% SDS-PAGE gel (Biorad, Hercules, CA). .. The membrane was then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 2 hr, followed by incubation with 0.1 μg/ml streptavidin-HRP conjugate (Pierce, Rockford, IL) in 1% BSA for 1 hr.

    Article Title: Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics
    Article Snippet: .. Captured proteins were eluted from the beads and subjected to SDS-PAGE and Western blot analysis using a streptavidin-HRP conjugate. ..

    Irradiation:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: HxBP (30 µM final concentration) or DMSO (vehicle control) was then added, and the solution was cross-linked using 48 mJ/cm2 of UV irradiation (Stratalinker 1800) (control tubes were left unexposed). .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Negative Control:

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿
    Article Snippet: The 2D blot probed with secondary antibody alone served as the negative control. .. Rickettsial surface proteins were detected by incubation of the membrane with streptavidin-HRP conjugate (Invitrogen) at a dilution of 1:6,000 for 1 h at room temperature.

    Recombinant:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: Paragraph title: In vitro Labeling of Human Recombinant MMP-2 ... The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis
    Article Snippet: Paragraph title: 4.1. Antibodies, Recombinant Proteins and Detection Agents ... Streptavidin-HRP conjugate was obtained from Thermo Scientific, Rockford, IL, USA.

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis
    Article Snippet: Screening of IL-17RA-Targeted Binders in ELISA Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 µg/mL, produced in E. coli SHuffle strain) or 5 µg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R & D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight. .. The samples of bacterial lysates in PBSTB or purified proteins were applied as a series of different dilutions in PBSTB and bound biotinylated ARS proteins were detected using streptavidin-HRP conjugate diluted in the same buffer 1:10,000 (Pierce, Rockford, IL, USA).

    In Vitro:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: Paragraph title: In vitro Labeling of Human Recombinant MMP-2 ... The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Radio Immunoprecipitation:

    Article Title: Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy
    Article Snippet: Cells were lysed in 0.3 mL radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors, and 0.01 mL of cell lysate (30 μg) was incubated with 100 ng of HA22 in ADP ribosylation buffer [20 mM Tris·HCl (pH 7.4), 1 mM EDTA, and 50 mM DTT] with 5 mM 6-Biotin-17-NAD (Trevigen) for 60 min at 25 °C. .. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate (Invitrogen) to detect biotin-ADP ribose-EF2.

    Activation Assay:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: In vitro Labeling of Human Recombinant MMP-2 Human recombinant MMP-2 was dissolved in activation buffer (50 mM NaCl, 50 mM Tris, 0.005% Triton X-100, pH 7.5) to a final concentration of 1 µg/ml in the presence or absence of 1 mM p -aminophenylmercuric acetate (APMA) and incubated 30 min at 37°C. .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Produced:

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis
    Article Snippet: Screening of IL-17RA-Targeted Binders in ELISA Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 µg/mL, produced in E. coli SHuffle strain) or 5 µg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R & D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight. .. The samples of bacterial lysates in PBSTB or purified proteins were applied as a series of different dilutions in PBSTB and bound biotinylated ARS proteins were detected using streptavidin-HRP conjugate diluted in the same buffer 1:10,000 (Pierce, Rockford, IL, USA).

    Concentration Assay:

    Article Title: Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    Article Snippet: HxBP (30 µM final concentration) or DMSO (vehicle control) was then added, and the solution was cross-linked using 48 mJ/cm2 of UV irradiation (Stratalinker 1800) (control tubes were left unexposed). .. The blot was incubated in blocking buffer (5% BSA in phosphate-buffered saline with 0.01% Tween-20 (PBSTw)) overnight at 4°C to prevent nonspecific binding, and then incubated in Streptavidin-HRP conjugate (Invitrogen) (diluted 1∶10000 in blocking buffer) overnight at 4°C.

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes
    Article Snippet: Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed. .. Other detection reagents (HRP-linked anti-biotin antibody from CST; strepavidin-HRP from R & D systems) were less sensitive, and higher concentration of the streptavidin-HRP conjugate resulted in higher background in empty wells.

    Staining:

    Article Title: Proteomic Analysis of Rickettsia parkeri Strain Portsmouth ▿
    Article Snippet: Rickettsial surface proteins were detected by incubation of the membrane with streptavidin-HRP conjugate (Invitrogen) at a dilution of 1:6,000 for 1 h at room temperature. .. The developed membranes were stained with an MemCode reversible protein stain kit (Pierce), according to the manufacturer's protocol, to match the location of proteins on the membrane with the Western blot signals.

    Gradient Centrifugation:

    Article Title: Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation
    Article Snippet: Then cells were lysed in lysis buffer and analyzed by sucrose gradient centrifugation and coimmunoprecipitation assays as described above. .. Biotinylated proteins were visualized with streptavidin-HRP conjugate ( Pierce Chemical Co. ) in conjunction with enhanced chemiluminescence ( Boehringer Mannheim , Indianapolis, IN).

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  • 93
    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: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
    Average 93 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated hrp - by Bioz Stars, 2020-04
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    97
    Thermo Fisher streptavidin hrp conjugates
    Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with <t>HRP-conjugated</t> <t>streptavidin,</t> and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.
    Streptavidin Hrp Conjugates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp conjugates/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp conjugates - by Bioz Stars, 2020-04
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    94
    Thermo Fisher streptavidin hrp conjugate
    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by <t>streptavidin-HRP</t> conjugate. Each point represents the mean value ± standard deviation (SD).
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp conjugate/product/Thermo Fisher
    Average 94 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp conjugate - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    Image Search Results


    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

    Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Journal: Scientific Reports

    Article Title: Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

    doi: 10.1038/s41598-018-20527-6

    Figure Lengend Snippet: Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Article Snippet: The entire blot was probed with streptavidin-HRP conjugates, and the entire image is displayed in each of the figures along with longer exposures of the region of the gel corresponding to the excised products of repair.

    Techniques: Irradiation, Incubation, Molecular Weight, Labeling, Nucleic Acid Electrophoresis, SDS Page, Mutagenesis

    Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Journal: International Journal of Molecular Sciences

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    doi: 10.3390/ijms19103089

    Figure Lengend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

    Article Snippet: Streptavidin-HRP conjugate was obtained from Thermo Scientific, Rockford, IL, USA.

    Techniques: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

    Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Journal: International Journal of Molecular Sciences

    Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

    doi: 10.3390/ijms19103089

    Figure Lengend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

    Article Snippet: Streptavidin-HRP conjugate was obtained from Thermo Scientific, Rockford, IL, USA.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo