streptavidin  (Millipore)


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  • 99
    Name:
    Anti Streptavidin antibody
    Description:

    Catalog Number:
    S6390
    Price:
    None
    Applications:
    Anti-Streptavidin antibody produced in rabbit was used in immunoblotting as a control to streptavidin-conjugated single chain antibody that was developed to bind Bacillus cereus spores.
    Host:
    rabbit
    Conjugate:
    unconjugated
    Immunogen:
    streptavidin from Streptomyces avidinii
    Category:
    Antibodies
    Preparation:
    Prepared using the periodate method described by Wilson M B and Nakane P K in Immunofluorescence and Related Staining Techniques Elsevier North Holland Biomedical Press Amsterdam p215 1978
    Source:
    goat
    Score:
    85
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    Structured Review

    Millipore streptavidin
    Biotin is not essential for survival of blood-stage P. falciparum . ( A ) Blood-stage P. falciparum cultures were maintained for 8 d in media with or without biotin. Four parasite lines were used: the parental control (Dd2), parasites overexpressing BCCP in the apicoplast under the ribosomal L2 (RL2) promoter, and parasites overexpressing BCCP in the cytosol under the RL2 promoter or the stronger CaM promoter. Error bars represent SEM of three biological replicates. ( B ) On day 8 of the growth assay shown in A , parasites were harvested and biotinylation levels were assessed by <t>streptavidin-HRP</t> affinity blotting, followed by Western blotting with αBCCP antibodies. There was no detectable biotinylated BCCP under biotin-free conditions, indicating that biotin was completely depleted in these cultures. Controls were parasites cultured in complete medium. ( C ) Blood-stage parasites (Dd2) were cultured with or without biotin for 7 d in media containing lipid-free BSA reconstituted with 30 µM palmitic acid and 30 µM oleic acid. Error bars represent SEM of three biological replicates. ( D ) TLC of parasites labeled with 14 C-acetate showing incorporation into C16 and C18 fatty acids. This activity was seen in WT and FASII KO parasites, and was independent of biotin. Similar results were obtained when this experiment was repeated ( Bottom ) using the minimal fatty acid medium described in C . No activity was seen with the negative control, uninfected red blood cells (RBC).

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    Images

    1) Product Images from "Host biotin is required for liver stage development in malaria parasites"

    Article Title: Host biotin is required for liver stage development in malaria parasites

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

    doi: 10.1073/pnas.1800717115

    Biotin is not essential for survival of blood-stage P. falciparum . ( A ) Blood-stage P. falciparum cultures were maintained for 8 d in media with or without biotin. Four parasite lines were used: the parental control (Dd2), parasites overexpressing BCCP in the apicoplast under the ribosomal L2 (RL2) promoter, and parasites overexpressing BCCP in the cytosol under the RL2 promoter or the stronger CaM promoter. Error bars represent SEM of three biological replicates. ( B ) On day 8 of the growth assay shown in A , parasites were harvested and biotinylation levels were assessed by streptavidin-HRP affinity blotting, followed by Western blotting with αBCCP antibodies. There was no detectable biotinylated BCCP under biotin-free conditions, indicating that biotin was completely depleted in these cultures. Controls were parasites cultured in complete medium. ( C ) Blood-stage parasites (Dd2) were cultured with or without biotin for 7 d in media containing lipid-free BSA reconstituted with 30 µM palmitic acid and 30 µM oleic acid. Error bars represent SEM of three biological replicates. ( D ) TLC of parasites labeled with 14 C-acetate showing incorporation into C16 and C18 fatty acids. This activity was seen in WT and FASII KO parasites, and was independent of biotin. Similar results were obtained when this experiment was repeated ( Bottom ) using the minimal fatty acid medium described in C . No activity was seen with the negative control, uninfected red blood cells (RBC).
    Figure Legend Snippet: Biotin is not essential for survival of blood-stage P. falciparum . ( A ) Blood-stage P. falciparum cultures were maintained for 8 d in media with or without biotin. Four parasite lines were used: the parental control (Dd2), parasites overexpressing BCCP in the apicoplast under the ribosomal L2 (RL2) promoter, and parasites overexpressing BCCP in the cytosol under the RL2 promoter or the stronger CaM promoter. Error bars represent SEM of three biological replicates. ( B ) On day 8 of the growth assay shown in A , parasites were harvested and biotinylation levels were assessed by streptavidin-HRP affinity blotting, followed by Western blotting with αBCCP antibodies. There was no detectable biotinylated BCCP under biotin-free conditions, indicating that biotin was completely depleted in these cultures. Controls were parasites cultured in complete medium. ( C ) Blood-stage parasites (Dd2) were cultured with or without biotin for 7 d in media containing lipid-free BSA reconstituted with 30 µM palmitic acid and 30 µM oleic acid. Error bars represent SEM of three biological replicates. ( D ) TLC of parasites labeled with 14 C-acetate showing incorporation into C16 and C18 fatty acids. This activity was seen in WT and FASII KO parasites, and was independent of biotin. Similar results were obtained when this experiment was repeated ( Bottom ) using the minimal fatty acid medium described in C . No activity was seen with the negative control, uninfected red blood cells (RBC).

    Techniques Used: Chick Chorioallantoic Membrane Assay, Growth Assay, Western Blot, Cell Culture, Thin Layer Chromatography, Labeling, Activity Assay, Gene Knockout, Negative Control

    Robust biotinylation activity can be detected in the cytosol but not the apicoplast of blood-stage P. falciparum . ( A ) The biotinylation domain (BCCP) from Pf ACC was expressed as a GFP fusion protein in the apicoplast of blood-stage P. falciparum by using the apicoplast targeting domain from Pf ACP (ACP-BCCP-GFP) under control of the ribosomal L2 (RL2) promoter. Localization was verified by epifluorescence microscopy in late ring ( Top ), trophozoite ( Middle ), and schizont ( Bottom ) parasites. Parasites were stained with MitoTracker to identify the mitochondrion and DAPI to identify nuclei. ( B ) BCCP was expressed as a GFP fusion protein in the cytosol (BCCP-GFP) under control of the ribosomal L2 (RL2) promoter, and localization was verified by epifluorescence microscopy as in A . ( C ) BCCP was expressed as a GFP fusion protein in the cytosol (BCCP-GFP) under control of the stronger CaM promoter, and localization was verified by epifluorescence microscopy as in A . ( D – F ) Whole-cell lysates from the parasite lines shown in A – C were analyzed by Western blotting with antibodies against GFP and affinity blotting with streptavidin-HRP. BCCP is biotinylated in the cytosol but is not biotinylated when expressed in the apicoplast. (Scale bars: 5 µm.)
    Figure Legend Snippet: Robust biotinylation activity can be detected in the cytosol but not the apicoplast of blood-stage P. falciparum . ( A ) The biotinylation domain (BCCP) from Pf ACC was expressed as a GFP fusion protein in the apicoplast of blood-stage P. falciparum by using the apicoplast targeting domain from Pf ACP (ACP-BCCP-GFP) under control of the ribosomal L2 (RL2) promoter. Localization was verified by epifluorescence microscopy in late ring ( Top ), trophozoite ( Middle ), and schizont ( Bottom ) parasites. Parasites were stained with MitoTracker to identify the mitochondrion and DAPI to identify nuclei. ( B ) BCCP was expressed as a GFP fusion protein in the cytosol (BCCP-GFP) under control of the ribosomal L2 (RL2) promoter, and localization was verified by epifluorescence microscopy as in A . ( C ) BCCP was expressed as a GFP fusion protein in the cytosol (BCCP-GFP) under control of the stronger CaM promoter, and localization was verified by epifluorescence microscopy as in A . ( D – F ) Whole-cell lysates from the parasite lines shown in A – C were analyzed by Western blotting with antibodies against GFP and affinity blotting with streptavidin-HRP. BCCP is biotinylated in the cytosol but is not biotinylated when expressed in the apicoplast. (Scale bars: 5 µm.)

    Techniques Used: Activity Assay, Epifluorescence Microscopy, Staining, Chick Chorioallantoic Membrane Assay, Western Blot

    2) Product Images from "Cargo Delivery into the Brain by in vivo identified Transport Peptides"

    Article Title: Cargo Delivery into the Brain by in vivo identified Transport Peptides

    Journal: Scientific Reports

    doi: 10.1038/srep14104

    Validation of CSF enriched phage-displayed peptides and CSF transport of biotinylated lead peptides conjugated to streptavidin payload. ( a ) Calculated enrichment factors based on injected (input = I) phage titers (PFU) and determined CSF phage titers (output = O) over all four rounds (R1-R4). The enrichment factors for the last three rounds (R2-R4) were calculated by the comparison with the previous round and the first round (R1) with the wt data. Open bars are CSF and stipple bars are plasma. (***p
    Figure Legend Snippet: Validation of CSF enriched phage-displayed peptides and CSF transport of biotinylated lead peptides conjugated to streptavidin payload. ( a ) Calculated enrichment factors based on injected (input = I) phage titers (PFU) and determined CSF phage titers (output = O) over all four rounds (R1-R4). The enrichment factors for the last three rounds (R2-R4) were calculated by the comparison with the previous round and the first round (R1) with the wt data. Open bars are CSF and stipple bars are plasma. (***p

    Techniques Used: Injection

    Lead transport peptide enhances brain BACE1 peptide inhibitory activity. ( a ) Long term CSF pharmacokinetic profiles shown for the clonally injected (2 × 10 10 phages/animal) T7 phage displayed #2077 (RLSSVDSDLSGC) peptide and the insert-less control phage (#1779) in at least three CM cannulated rats each. ( b ) Confocal microscopic image of a representative cortex microvessel in a rat i.v. injected with phage (2 × 10 10 phages/animal) displaying the #2077 peptide and a vascular counterstaining (lectin). Indicated phage clones were injected into 3 rats and allowed to circulate for 1 hour before perfusion. The brain sectioned and stained with a polyclonal FITC labeled antibody against the T7 phage capsid. 10 minutes before perfusion and subsequent fixation DyLight594 labeled lectin was i.v. injected. Fluorescence images showing lectin (red) stained luminal side of the microvessel and the phage (green) in the capillary lumen and the perivascular brain tissue. Scale bar corresponds to 10 μm. ( c,d ) A biotinylated BACE1 inhibitory peptide alone or in combination with the biotinylated #2077 transport peptide was attached to streptavidin and subsequently i.v. injected (10 mg streptavidin/kg) in at least three CM cannulated rats each. BACE1 peptide inhibitor mediated Aβ40 reduction was measured by an Aβ1-40 ELISA in blood (red) and CSF (orange) at the indicated time points. For better visibility, a dashed line at 100% was drawn in the graphs. ( c ) The percentage blood (red triangles) and CSF (orange triangles) Aβ40 reduction in rats injected with streptavidin attached to the #2077 transport peptide and the BACE1 inhibitor peptide at a 3:1 ratio. ( d ) The percentage blood (red circles) and CSF (orange circles) Aβ40 reduction in rats injected with streptavidin attached with only BACE1 inhibitor peptides. The Aβ concentration for the control was 420 pg/ml (Std. deviation = 101 pg/ml).
    Figure Legend Snippet: Lead transport peptide enhances brain BACE1 peptide inhibitory activity. ( a ) Long term CSF pharmacokinetic profiles shown for the clonally injected (2 × 10 10 phages/animal) T7 phage displayed #2077 (RLSSVDSDLSGC) peptide and the insert-less control phage (#1779) in at least three CM cannulated rats each. ( b ) Confocal microscopic image of a representative cortex microvessel in a rat i.v. injected with phage (2 × 10 10 phages/animal) displaying the #2077 peptide and a vascular counterstaining (lectin). Indicated phage clones were injected into 3 rats and allowed to circulate for 1 hour before perfusion. The brain sectioned and stained with a polyclonal FITC labeled antibody against the T7 phage capsid. 10 minutes before perfusion and subsequent fixation DyLight594 labeled lectin was i.v. injected. Fluorescence images showing lectin (red) stained luminal side of the microvessel and the phage (green) in the capillary lumen and the perivascular brain tissue. Scale bar corresponds to 10 μm. ( c,d ) A biotinylated BACE1 inhibitory peptide alone or in combination with the biotinylated #2077 transport peptide was attached to streptavidin and subsequently i.v. injected (10 mg streptavidin/kg) in at least three CM cannulated rats each. BACE1 peptide inhibitor mediated Aβ40 reduction was measured by an Aβ1-40 ELISA in blood (red) and CSF (orange) at the indicated time points. For better visibility, a dashed line at 100% was drawn in the graphs. ( c ) The percentage blood (red triangles) and CSF (orange triangles) Aβ40 reduction in rats injected with streptavidin attached to the #2077 transport peptide and the BACE1 inhibitor peptide at a 3:1 ratio. ( d ) The percentage blood (red circles) and CSF (orange circles) Aβ40 reduction in rats injected with streptavidin attached with only BACE1 inhibitor peptides. The Aβ concentration for the control was 420 pg/ml (Std. deviation = 101 pg/ml).

    Techniques Used: Activity Assay, Injection, Clone Assay, Staining, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay

    3) Product Images from "Translational profiling through biotinylation of tagged ribosomes in zebrafish"

    Article Title: Translational profiling through biotinylation of tagged ribosomes in zebrafish

    Journal:

    doi: 10.1242/dev.111849

    Specific biotinylation of Avi-tagged EGFP in zebrafish embryos. (A) Avi-tagged EGFP (arrowhead) is selectively biotinylated when co-expressed with BirA, as shown by SDS-PAGE and streptavidin-HRP blotting (upper panel) of lysates from zebrafish embryos transiently expressing Avi-EGFP and BirA. The middle panel is an anti-GFP antibody blot confirming expression of Avi-EGFP. The bottom panel is an anti-β-tubulin (Tubb) antibody blot shown as a loading control. BirA and Avi-EGFP were transiently expressed by co-injection of 500 pg of in vitro transcribed mRNA into one-cell stage zebrafish embryos. Lysates for blotting analysis were generated from 24 hpf embryos. (B) Streptavidin gel shift assay (upper panel, anti-GFP antibody blot) performed on zebrafish embryos treated with indicated amounts of exogenous biotin following transient expression of Avi-EGFP and BirA. The lower panel is a streptavidin-HRP blot of the endogenously biotinylated 130 kDa protein shown as a loading control. Transient expression was performed as in A. (C) SDS-PAGE and Coomassie Blue G250 staining of streptavidin-agarose purified Avi-EGFP from lysates generated from 50 zebrafish embryos transiently expressing Avi-EGFP and BirA. Lane 2 contains 50 ng of BSA for comparison. Transient expression was performed by mRNA injection as in A. Thin lines in B,C mark positions where lanes from the same gel have been spliced together.
    Figure Legend Snippet: Specific biotinylation of Avi-tagged EGFP in zebrafish embryos. (A) Avi-tagged EGFP (arrowhead) is selectively biotinylated when co-expressed with BirA, as shown by SDS-PAGE and streptavidin-HRP blotting (upper panel) of lysates from zebrafish embryos transiently expressing Avi-EGFP and BirA. The middle panel is an anti-GFP antibody blot confirming expression of Avi-EGFP. The bottom panel is an anti-β-tubulin (Tubb) antibody blot shown as a loading control. BirA and Avi-EGFP were transiently expressed by co-injection of 500 pg of in vitro transcribed mRNA into one-cell stage zebrafish embryos. Lysates for blotting analysis were generated from 24 hpf embryos. (B) Streptavidin gel shift assay (upper panel, anti-GFP antibody blot) performed on zebrafish embryos treated with indicated amounts of exogenous biotin following transient expression of Avi-EGFP and BirA. The lower panel is a streptavidin-HRP blot of the endogenously biotinylated 130 kDa protein shown as a loading control. Transient expression was performed as in A. (C) SDS-PAGE and Coomassie Blue G250 staining of streptavidin-agarose purified Avi-EGFP from lysates generated from 50 zebrafish embryos transiently expressing Avi-EGFP and BirA. Lane 2 contains 50 ng of BSA for comparison. Transient expression was performed by mRNA injection as in A. Thin lines in B,C mark positions where lanes from the same gel have been spliced together.

    Techniques Used: SDS Page, Expressing, Injection, In Vitro, Generated, Electrophoretic Mobility Shift Assay, Staining, Purification

    4) Product Images from "Analysis of the Nse3/MAGE-Binding Domain of the Nse4/EID Family Proteins"

    Article Title: Analysis of the Nse3/MAGE-Binding Domain of the Nse4/EID Family Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035813

    Analysis of EID2 binding to MAGE proteins. ( A. ) Quantification of relative binding of the MAGEC2(129-339) protein (red columns) to the EID2 protein-based synthetic mutant peptides (listed in Table 1 ) using the PEPSCAN-ELISA method. Results show mean ± SEM of 3 independent measurements. His-hTRF2 protein (white column) was used in the control experiment. ( B. to D. ) The short (biotin-SGSG- 201 HRVDLDILTFTIALTAS 217 ) and long (biotin-SGSG- 197 QRNPHRVDLDILTFTIALTAS 217 ) EID2 peptides were pre-bound to the streptavidin-agarose beads and then incubated with in vitro translated MAGEC2 (aa 6-373; C2 in panel B. ), MAGEA1 (aa 1-309; A1 in panel C. ) and/or necdin (aa 1-321; nd in panel D. ) protein, respectively. ( E. ) Wild type and selected EID2 mutant peptides (as indicated) were pre-bound to the streptavidin-agarose beads and then incubated with in vitro translated necdin protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the in vitro translated proteins was measured by autoradiography. Control, no peptide.
    Figure Legend Snippet: Analysis of EID2 binding to MAGE proteins. ( A. ) Quantification of relative binding of the MAGEC2(129-339) protein (red columns) to the EID2 protein-based synthetic mutant peptides (listed in Table 1 ) using the PEPSCAN-ELISA method. Results show mean ± SEM of 3 independent measurements. His-hTRF2 protein (white column) was used in the control experiment. ( B. to D. ) The short (biotin-SGSG- 201 HRVDLDILTFTIALTAS 217 ) and long (biotin-SGSG- 197 QRNPHRVDLDILTFTIALTAS 217 ) EID2 peptides were pre-bound to the streptavidin-agarose beads and then incubated with in vitro translated MAGEC2 (aa 6-373; C2 in panel B. ), MAGEA1 (aa 1-309; A1 in panel C. ) and/or necdin (aa 1-321; nd in panel D. ) protein, respectively. ( E. ) Wild type and selected EID2 mutant peptides (as indicated) were pre-bound to the streptavidin-agarose beads and then incubated with in vitro translated necdin protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the in vitro translated proteins was measured by autoradiography. Control, no peptide.

    Techniques Used: Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Incubation, In Vitro, SDS Page, Nucleic Acid Electrophoresis, Autoradiography

    5) Product Images from "Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement"

    Article Title: Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07726-5

    Bioconjugation strategy for cell surface labelling using AuNP. a Explanatory sketch illustrating the structure of the pH nanosensor: the surface of AuNP is functionalized with 4-MBA and HPDP-B. The sulfo-NHS moiety of the biotinylation reagent NHS-B reacts with primary amines in lysines or amino-termini of membrane proteins such as receptors, pores, channels, carriers, pumps, integrins, or enzymes. Two of the four active sites of streptavidin (SA) provide the link between the biotins of NHS-B anchored to proteins and HPDP-B conjugated to AuNP. b Conjugated AuNP labelled with Alexa-SA: the attachment of the nanoparticles to the cell membrane was confirmed using AuNP functionalized with HPDP-B, some biotins of which reacted with Alexa-SA to enable visualization by CFM
    Figure Legend Snippet: Bioconjugation strategy for cell surface labelling using AuNP. a Explanatory sketch illustrating the structure of the pH nanosensor: the surface of AuNP is functionalized with 4-MBA and HPDP-B. The sulfo-NHS moiety of the biotinylation reagent NHS-B reacts with primary amines in lysines or amino-termini of membrane proteins such as receptors, pores, channels, carriers, pumps, integrins, or enzymes. Two of the four active sites of streptavidin (SA) provide the link between the biotins of NHS-B anchored to proteins and HPDP-B conjugated to AuNP. b Conjugated AuNP labelled with Alexa-SA: the attachment of the nanoparticles to the cell membrane was confirmed using AuNP functionalized with HPDP-B, some biotins of which reacted with Alexa-SA to enable visualization by CFM

    Techniques Used:

    6) Product Images from "Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair"

    Article Title: Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

    Journal:

    doi: 10.1093/emboj/cdg603

    Fig. 1. The extreme C-terminus of the α-subunit of DNA polymerase III interacts with the β-clamp. ( A ) Sequences probed by peptide analysis are shown as stippled boxes in the DnaE protein scheme, but only the analysis of the C-terminal peptides is shown. N-terminal biotinylated peptides were immobilized on streptavidin-coated 96-well plates and probed with 32 P-β. It is estimated that 30 nmol of peptide is retained in each well. Peptide sequences used in the microplate assay on the right are shown as lines under the sequence of the C-terminus. Results of the assays are shown to the right. ( B ) Native polyacrylamide electrophoresis was used to separate a complex of 32 P-β with biotinylated α1141–1160 bound to streptavidin. Lane 1 contained only 32 P-β; the α1141–1160 peptide (220 nM) was added in lanes 3 and 4, and streptavidin (2.2 µM) in lanes 2 and 4, as indicated. ( C ) Gel shift assay of the radiolabeled β-clamp. Native polyacrylamide electrophoresis was performed to separate free 32 P-β from 32 P-β·core complexes as described in Materials and methods. Pol III core was added in lanes 2–6 and the complex was challenged with either BSA (lane 2), non-labeled E.coli β-clamp (lane 3), human PCNA (lane 4), phage T4 gp45 (lane 5) or α1141–1160 peptide (lane 6), as indicated above the gel. ( D ) DNA synthesis is inhibited by α C-terminal peptides. Reactions were performed using primed M13mp18 ssDNA, β, core and γ complex in the presence of 100 µM of each peptide as described in Materials and methods.
    Figure Legend Snippet: Fig. 1. The extreme C-terminus of the α-subunit of DNA polymerase III interacts with the β-clamp. ( A ) Sequences probed by peptide analysis are shown as stippled boxes in the DnaE protein scheme, but only the analysis of the C-terminal peptides is shown. N-terminal biotinylated peptides were immobilized on streptavidin-coated 96-well plates and probed with 32 P-β. It is estimated that 30 nmol of peptide is retained in each well. Peptide sequences used in the microplate assay on the right are shown as lines under the sequence of the C-terminus. Results of the assays are shown to the right. ( B ) Native polyacrylamide electrophoresis was used to separate a complex of 32 P-β with biotinylated α1141–1160 bound to streptavidin. Lane 1 contained only 32 P-β; the α1141–1160 peptide (220 nM) was added in lanes 3 and 4, and streptavidin (2.2 µM) in lanes 2 and 4, as indicated. ( C ) Gel shift assay of the radiolabeled β-clamp. Native polyacrylamide electrophoresis was performed to separate free 32 P-β from 32 P-β·core complexes as described in Materials and methods. Pol III core was added in lanes 2–6 and the complex was challenged with either BSA (lane 2), non-labeled E.coli β-clamp (lane 3), human PCNA (lane 4), phage T4 gp45 (lane 5) or α1141–1160 peptide (lane 6), as indicated above the gel. ( D ) DNA synthesis is inhibited by α C-terminal peptides. Reactions were performed using primed M13mp18 ssDNA, β, core and γ complex in the presence of 100 µM of each peptide as described in Materials and methods.

    Techniques Used: Sequencing, Electrophoresis, Electrophoretic Mobility Shift Assay, Labeling, DNA Synthesis

    Fig. 2. Alanine scan analysis of α C-terminal peptide binding to β. ( A ) Peptides were pre-bound to streptavidin-coated microtiter plates and probed with 32 P-β as in Figure A. A clear spot indicates a critical residue for 32 P-β binding. ( B ) Peptides were used to compete Pol III core off the 32 P-β·core complex in the native PAGE mobility shift assay, as in Figure C. ( C ) Purified δ protein (20 pmol) was fixed to microtiter plates and probed with 32 P-β in the presence of the indicated peptide. Retention of 32 P-β in the well indicates that a critical residue has been changed to Ala.
    Figure Legend Snippet: Fig. 2. Alanine scan analysis of α C-terminal peptide binding to β. ( A ) Peptides were pre-bound to streptavidin-coated microtiter plates and probed with 32 P-β as in Figure A. A clear spot indicates a critical residue for 32 P-β binding. ( B ) Peptides were used to compete Pol III core off the 32 P-β·core complex in the native PAGE mobility shift assay, as in Figure C. ( C ) Purified δ protein (20 pmol) was fixed to microtiter plates and probed with 32 P-β in the presence of the indicated peptide. Retention of 32 P-β in the well indicates that a critical residue has been changed to Ala.

    Techniques Used: Binding Assay, Clear Native PAGE, Mobility Shift, Purification

    7) Product Images from "Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3? to 5? translocase and helicase activities"

    Article Title: Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3? to 5? translocase and helicase activities

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks417

    Directionality of SftH translocation on ssDNA. Translocase assays were performed as described under Methods. Native PAGE analysis of the reaction products are shown. The species corresponding to SA–DNA complex and free DNA are indicated. The nucleobase sequences of the 5 ′ or 3 ′ biotinylated 34-mer single-stranded DNAs are shown at bottom with ( B ) signifying the position of the biotin spacer. The complete translocase reaction mixtures contained 1 mM ATP (+), 5 mM MgCl 2 (+), 0.5 pmol 32 P-labeled 3 ′ or 5 ′ biotinylated DNA attached to streptavidin (+), and 1 pmol of either wild-type SftH (+ or WT) or mutants K190A or D195A as specified. Streptavidin, ATP, magnesium and/or SftH were omitted (−) from control reactions as indicated above the lanes. The reactions in panel A were performed with the 5′ biotinylated or 3' biotinylated DNAs as indicated. All of the reactions in panel B were performed with the 5 ′ biotinylated DNA.
    Figure Legend Snippet: Directionality of SftH translocation on ssDNA. Translocase assays were performed as described under Methods. Native PAGE analysis of the reaction products are shown. The species corresponding to SA–DNA complex and free DNA are indicated. The nucleobase sequences of the 5 ′ or 3 ′ biotinylated 34-mer single-stranded DNAs are shown at bottom with ( B ) signifying the position of the biotin spacer. The complete translocase reaction mixtures contained 1 mM ATP (+), 5 mM MgCl 2 (+), 0.5 pmol 32 P-labeled 3 ′ or 5 ′ biotinylated DNA attached to streptavidin (+), and 1 pmol of either wild-type SftH (+ or WT) or mutants K190A or D195A as specified. Streptavidin, ATP, magnesium and/or SftH were omitted (−) from control reactions as indicated above the lanes. The reactions in panel A were performed with the 5′ biotinylated or 3' biotinylated DNAs as indicated. All of the reactions in panel B were performed with the 5 ′ biotinylated DNA.

    Techniques Used: Translocation Assay, Clear Native PAGE, Labeling

    8) Product Images from "A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction"

    Article Title: A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction

    Journal: Glycobiology

    doi: 10.1093/glycob/cwp085

    Efficacy of clusterization and antibody response. BALB/c mouse ( n = 6) sera were assayed after the third Tn-cBSA immunization. The histogram reports the mean values ± standard deviation (SD) of a comparative ELISA performed on Tn clusterized on alginate (Tn-Alg), alginate (Alg), and Tn (GalNAc-α- O -Ser). Mouse serum was used at a dilution of 1:100. Tn-alginate conjugation significantly improved the binding of specific antibodies to the target antigen compared with unclusterized Tn ( * P = 0.01). For these experiments, all the compounds were biotinylated to assure their immobilization on a streptavidin-coated 96-well plate and a polyvalent IgG/A/M-HRP secondary antibody was used. OD, optical density.
    Figure Legend Snippet: Efficacy of clusterization and antibody response. BALB/c mouse ( n = 6) sera were assayed after the third Tn-cBSA immunization. The histogram reports the mean values ± standard deviation (SD) of a comparative ELISA performed on Tn clusterized on alginate (Tn-Alg), alginate (Alg), and Tn (GalNAc-α- O -Ser). Mouse serum was used at a dilution of 1:100. Tn-alginate conjugation significantly improved the binding of specific antibodies to the target antigen compared with unclusterized Tn ( * P = 0.01). For these experiments, all the compounds were biotinylated to assure their immobilization on a streptavidin-coated 96-well plate and a polyvalent IgG/A/M-HRP secondary antibody was used. OD, optical density.

    Techniques Used: Standard Deviation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Binding Assay

    9) Product Images from "A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction"

    Article Title: A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction

    Journal: Glycobiology

    doi: 10.1093/glycob/cwp085

    Efficacy of clusterization and antibody response. BALB/c mouse ( n = 6) sera were assayed after the third Tn-cBSA immunization. The histogram reports the mean values ± standard deviation (SD) of a comparative ELISA performed on Tn clusterized on alginate (Tn-Alg), alginate (Alg), and Tn (GalNAc-α- O -Ser). Mouse serum was used at a dilution of 1:100. Tn-alginate conjugation significantly improved the binding of specific antibodies to the target antigen compared with unclusterized Tn ( * P = 0.01). For these experiments, all the compounds were biotinylated to assure their immobilization on a streptavidin-coated 96-well plate and a polyvalent IgG/A/M-HRP secondary antibody was used. OD, optical density.
    Figure Legend Snippet: Efficacy of clusterization and antibody response. BALB/c mouse ( n = 6) sera were assayed after the third Tn-cBSA immunization. The histogram reports the mean values ± standard deviation (SD) of a comparative ELISA performed on Tn clusterized on alginate (Tn-Alg), alginate (Alg), and Tn (GalNAc-α- O -Ser). Mouse serum was used at a dilution of 1:100. Tn-alginate conjugation significantly improved the binding of specific antibodies to the target antigen compared with unclusterized Tn ( * P = 0.01). For these experiments, all the compounds were biotinylated to assure their immobilization on a streptavidin-coated 96-well plate and a polyvalent IgG/A/M-HRP secondary antibody was used. OD, optical density.

    Techniques Used: Standard Deviation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Binding Assay

    10) Product Images from "A potent human neutralizing antibody Fc-dependently reduces established HBV infections"

    Article Title: A potent human neutralizing antibody Fc-dependently reduces established HBV infections

    Journal: eLife

    doi: 10.7554/eLife.26738

    preS1 binding activity and neutralization activities of the top four nAbs obtained from 2H5-VH chain shuffling. ( A ) Evaluating the binding activities of nAbs to preS1 peptide. The nAbs in their full-length human IgG1 forms were tested for binding to 7.8 nM NC36b peptides captured on an ELISA plate that had been pre-coated with streptavidin. ( B-C ) Neutralization of HBV and HDV by nAbs. The percentage of HBeAg inhibition at dpi seven is shown in panel ( B ); the staining of HDV delta antigens at seven dpi is shown in panel ( C ).
    Figure Legend Snippet: preS1 binding activity and neutralization activities of the top four nAbs obtained from 2H5-VH chain shuffling. ( A ) Evaluating the binding activities of nAbs to preS1 peptide. The nAbs in their full-length human IgG1 forms were tested for binding to 7.8 nM NC36b peptides captured on an ELISA plate that had been pre-coated with streptavidin. ( B-C ) Neutralization of HBV and HDV by nAbs. The percentage of HBeAg inhibition at dpi seven is shown in panel ( B ); the staining of HDV delta antigens at seven dpi is shown in panel ( C ).

    Techniques Used: Binding Assay, Activity Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Inhibition, Staining

    Binding of the six anti-preS1 nAbs to preS1 peptides and characterization of their epitopes. ( A ) Binding of anti-preS1 nAbs to preS1 peptides by ELISA. The selected six Abs in their full-length human IgG1 forms were tested for binding to 188 nM preS1 peptides captured on an ELISA plate that had been pre-coated with streptavidin. 2H5, m1Q, and T47, bound to all three of the peptides. #71, and #76 bound to NC36b and 47b but not m47b. #15 bound to m47b and 47b, but not NC36b. The binding of Abs was detected by HRP-anti-human Fc secondary Ab. ( B ) ELISA-based assay of Abs binding to the shorter preS1 peptides, NN23b and LD23b. The assay was performed as in panel A. ( C ) Competition ELISA with short preS1 peptides. In the presence of competition short peptides without biotinylation at the indicated concentrations, the binding activity of Abs with 188 nM biotinylated m47b or 47b preS1 peptide was measured. ( A–C ) The different epitopes recognized by the nAbs are depicted in Figure 1—figure supplement 1 . ( D ) Competition ELISA with mutant peptides. Amino acids of preS1, Leu 19 , Asp 20 , Pro 21 , or Phe 23 were mutated to alanine in LN16 (a 16-mer pre S1 peptide) and tested for competition activity against the binding of 2H5 to m47b; the assay was performed as in panel C. ( E ) G24R mutation in preS1 did not affect 2H5 binding.
    Figure Legend Snippet: Binding of the six anti-preS1 nAbs to preS1 peptides and characterization of their epitopes. ( A ) Binding of anti-preS1 nAbs to preS1 peptides by ELISA. The selected six Abs in their full-length human IgG1 forms were tested for binding to 188 nM preS1 peptides captured on an ELISA plate that had been pre-coated with streptavidin. 2H5, m1Q, and T47, bound to all three of the peptides. #71, and #76 bound to NC36b and 47b but not m47b. #15 bound to m47b and 47b, but not NC36b. The binding of Abs was detected by HRP-anti-human Fc secondary Ab. ( B ) ELISA-based assay of Abs binding to the shorter preS1 peptides, NN23b and LD23b. The assay was performed as in panel A. ( C ) Competition ELISA with short preS1 peptides. In the presence of competition short peptides without biotinylation at the indicated concentrations, the binding activity of Abs with 188 nM biotinylated m47b or 47b preS1 peptide was measured. ( A–C ) The different epitopes recognized by the nAbs are depicted in Figure 1—figure supplement 1 . ( D ) Competition ELISA with mutant peptides. Amino acids of preS1, Leu 19 , Asp 20 , Pro 21 , or Phe 23 were mutated to alanine in LN16 (a 16-mer pre S1 peptide) and tested for competition activity against the binding of 2H5 to m47b; the assay was performed as in panel C. ( E ) G24R mutation in preS1 did not affect 2H5 binding.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activity Assay, Mutagenesis

    11) Product Images from "In Vivo Delivery of Antisense MORF Oligomer by MORF/Carrier Streptavidin Nanoparticles"

    Article Title: In Vivo Delivery of Antisense MORF Oligomer by MORF/Carrier Streptavidin Nanoparticles

    Journal:

    doi: 10.1089/cbr.2009.0624

    Single-photon emission computed tomography/computed tomography images of a normal mouse at 30, 60, and 90 minutes postadministration of the 99m Tc-MORF/streptavidin/tat nanoparticle.
    Figure Legend Snippet: Single-photon emission computed tomography/computed tomography images of a normal mouse at 30, 60, and 90 minutes postadministration of the 99m Tc-MORF/streptavidin/tat nanoparticle.

    Techniques Used: Single Photon Emission Computed Tomography, Computed Tomography

    Radioactivity profiles by size-exclusion high-performance liquid chromatography. ( A ) 99m Tc-labeled antisense phosphorodiamidate morpholino (MORF). ( B ) 99m Tc-labeled antisense MORF/streptavidin nanoparticle before purification. ( C ) 99m Tc-labeled antisense
    Figure Legend Snippet: Radioactivity profiles by size-exclusion high-performance liquid chromatography. ( A ) 99m Tc-labeled antisense phosphorodiamidate morpholino (MORF). ( B ) 99m Tc-labeled antisense MORF/streptavidin nanoparticle before purification. ( C ) 99m Tc-labeled antisense

    Techniques Used: Radioactivity, High Performance Liquid Chromatography, Labeling, Purification

    12) Product Images from "Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy"

    Article Title: Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy

    Journal:

    doi: 10.1016/j.cplett.2010.02.053

    Raman spectra of 10−5 M streptavidin in water. The PDMS microfluidic channel was placed on either glass or, tilted and rotated AAO substrates. The background of water filled MFC, tilted at the respective angle was subtracted from the curves except for the data for glass. The best data was obtained for θ=0° (solid black curve). Characteristic signatures of streptavidin at 1410 cm−1 are assigned to C-H stretching of δCH2 , δCH3 . The 1228 cm−1 peak is assigned to N-H stretching of the β -sheet backbone amide bonds .
    Figure Legend Snippet: Raman spectra of 10−5 M streptavidin in water. The PDMS microfluidic channel was placed on either glass or, tilted and rotated AAO substrates. The background of water filled MFC, tilted at the respective angle was subtracted from the curves except for the data for glass. The best data was obtained for θ=0° (solid black curve). Characteristic signatures of streptavidin at 1410 cm−1 are assigned to C-H stretching of δCH2 , δCH3 . The 1228 cm−1 peak is assigned to N-H stretching of the β -sheet backbone amide bonds .

    Techniques Used:

    (a) Raman spectra of 10−5 M streptavidin injected into MFC on AAO substrate. The data was taken at normal incidence (tilt angle θ=0°). (a) Bare AAO substrate and (b) AAO substrate impregnated with DMPC. The streptavidin could be easily washed away from either substrate as noted by the flat curves after wash. The background spectrum of AAO with only DMPC was subtracted from each data.
    Figure Legend Snippet: (a) Raman spectra of 10−5 M streptavidin injected into MFC on AAO substrate. The data was taken at normal incidence (tilt angle θ=0°). (a) Bare AAO substrate and (b) AAO substrate impregnated with DMPC. The streptavidin could be easily washed away from either substrate as noted by the flat curves after wash. The background spectrum of AAO with only DMPC was subtracted from each data.

    Techniques Used: Injection

    13) Product Images from "4?-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo"

    Article Title: 4?-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003097

    In vitro activity of PptT. A. Diagrammatic representation of the domain organization of PKS13 and of ACP and ACPb domains. ACP: Acyl Carrier protein, KS: ketosynthase, AT: acyltransferase, TE: thioesterase. B. ACP activation with CoA and acetyl-CoA. The apo -ACP module was incubated with (+) or without (−) PptT in the presence of either CoA (left panel) or acetyl-CoA (right panel). apo - (a) and holo -ACP (h) forms were separated on urea polyacrylamide gels and stained with Coomassie blue (see materials and methods ). M: PageRuler prestained protein ladder plus (Fermentas). C. ACP activation with CoA analogs. PptT or Sfp were incubated with the apo -ACP domain in the presence of either fluorescent CoA analogs (CoA488 or CoA547) or CoA-biotin. Fluorescent holo -ACP forms (h) were resolved by SDS-PAGE and visualized by fluorescence scanning using a Typhoon scanner (GE Healthcare) (upper panel). Biotin-labeled ACP was detected by spotting 5 µl of the reaction mix onto the nitrocellulose membrane and incubation with streptavidin peroxidase followed by enhanced chemiluminescence detection (lower panel). The dashed-line circle shows the drop zone for the PptT reaction.
    Figure Legend Snippet: In vitro activity of PptT. A. Diagrammatic representation of the domain organization of PKS13 and of ACP and ACPb domains. ACP: Acyl Carrier protein, KS: ketosynthase, AT: acyltransferase, TE: thioesterase. B. ACP activation with CoA and acetyl-CoA. The apo -ACP module was incubated with (+) or without (−) PptT in the presence of either CoA (left panel) or acetyl-CoA (right panel). apo - (a) and holo -ACP (h) forms were separated on urea polyacrylamide gels and stained with Coomassie blue (see materials and methods ). M: PageRuler prestained protein ladder plus (Fermentas). C. ACP activation with CoA analogs. PptT or Sfp were incubated with the apo -ACP domain in the presence of either fluorescent CoA analogs (CoA488 or CoA547) or CoA-biotin. Fluorescent holo -ACP forms (h) were resolved by SDS-PAGE and visualized by fluorescence scanning using a Typhoon scanner (GE Healthcare) (upper panel). Biotin-labeled ACP was detected by spotting 5 µl of the reaction mix onto the nitrocellulose membrane and incubation with streptavidin peroxidase followed by enhanced chemiluminescence detection (lower panel). The dashed-line circle shows the drop zone for the PptT reaction.

    Techniques Used: In Vitro, Activity Assay, Activation Assay, Incubation, Staining, SDS Page, Fluorescence, Labeling

    14) Product Images from "EXD2 promotes homologous recombination by facilitating DNA-end resection"

    Article Title: EXD2 promotes homologous recombination by facilitating DNA-end resection

    Journal: Nature cell biology

    doi: 10.1038/ncb3303

    EXD2 displays 3′ – 5′ exonuclease activity in vitro a) 5′ radiolabeled ssDNA 50-mer substrate (10 nM molecules) was incubated for the indicated amounts of time with EXD2 WT or EXD2 D108A E110A mutant protein (70 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. b) 3′ radiolabeled ssDNA 50-mer substrate (0.25 μM molecules) was incubated for the indicated amounts of time with EXD2 WT or EXD2 D108A E110A (70 nM) mutant protein. Samples were resolved by TLC in 1M sodium formate pH 3.4 and visualised by phosphorimaging. This experiment was carried out two times independently. c) 5′ dsDNA 50-mer substrates (10 nM molecules) were incubated for the indicated amounts of time with EXD2 WT protein (70 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. d) 5′ radiolabeled ssDNA or dsDNA with 5’overhang substrate (3 nM molecules) was incubated for indicated amounts of time with EXD2 WT (K76 - V564) protein (25 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. e) 5′ radiolabeled ssDNA or dsDNA (3 nM molecules) with 3′ end blocked by biotin – streptavidin was incubated for indicated time with EXD2 WT (K76 - V564) protein (25 nM) in buffer supplemented with 1 mM ATP. Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. f) (upper panel) EXD2 WT (K76 - V564) gel-filtration fractions were tested for nuclease activity against 5′ radiolabeled ssDNA (10 nM molecules). Reactions were incubated for 30 min and resolved on a 15% TBE-Urea polyacrylamide gel and visualised by phosphorimaging; (lower panel) Coomassie blue–stained gel depicting the EXD2 protein in gel-filtration fractions analysed in the upper panel. This experiment was carried out two times independently.
    Figure Legend Snippet: EXD2 displays 3′ – 5′ exonuclease activity in vitro a) 5′ radiolabeled ssDNA 50-mer substrate (10 nM molecules) was incubated for the indicated amounts of time with EXD2 WT or EXD2 D108A E110A mutant protein (70 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. b) 3′ radiolabeled ssDNA 50-mer substrate (0.25 μM molecules) was incubated for the indicated amounts of time with EXD2 WT or EXD2 D108A E110A (70 nM) mutant protein. Samples were resolved by TLC in 1M sodium formate pH 3.4 and visualised by phosphorimaging. This experiment was carried out two times independently. c) 5′ dsDNA 50-mer substrates (10 nM molecules) were incubated for the indicated amounts of time with EXD2 WT protein (70 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. d) 5′ radiolabeled ssDNA or dsDNA with 5’overhang substrate (3 nM molecules) was incubated for indicated amounts of time with EXD2 WT (K76 - V564) protein (25 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. e) 5′ radiolabeled ssDNA or dsDNA (3 nM molecules) with 3′ end blocked by biotin – streptavidin was incubated for indicated time with EXD2 WT (K76 - V564) protein (25 nM) in buffer supplemented with 1 mM ATP. Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently. f) (upper panel) EXD2 WT (K76 - V564) gel-filtration fractions were tested for nuclease activity against 5′ radiolabeled ssDNA (10 nM molecules). Reactions were incubated for 30 min and resolved on a 15% TBE-Urea polyacrylamide gel and visualised by phosphorimaging; (lower panel) Coomassie blue–stained gel depicting the EXD2 protein in gel-filtration fractions analysed in the upper panel. This experiment was carried out two times independently.

    Techniques Used: Activity Assay, In Vitro, Incubation, Mutagenesis, Thin Layer Chromatography, Filtration, Staining

    15) Product Images from "Anomalous Diffusion of Proteins Due to Molecular Crowding"

    Article Title: Anomalous Diffusion of Proteins Due to Molecular Crowding

    Journal:

    doi: 10.1529/biophysj.104.051078

    ( a ) Anomalous diffusion exponent associated with the diffusion of streptavidin as a function of obstacle concentration for dextrans of various average molecular weights. Lines are fits to the data using Eq. 10 with α l = 0.74. Where necessary,
    Figure Legend Snippet: ( a ) Anomalous diffusion exponent associated with the diffusion of streptavidin as a function of obstacle concentration for dextrans of various average molecular weights. Lines are fits to the data using Eq. 10 with α l = 0.74. Where necessary,

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effective distributions in average residence times calculated with the MEMFCS algorithm for the experimental autocorrelation data shown in ( symbols ), and for two sets of simulated autocorrelation data corresponding to the case of streptavidin
    Figure Legend Snippet: Effective distributions in average residence times calculated with the MEMFCS algorithm for the experimental autocorrelation data shown in ( symbols ), and for two sets of simulated autocorrelation data corresponding to the case of streptavidin

    Techniques Used:

    Anomalous diffusion exponent as a function of dextran concentration fitted to Eq. 10 for various tracers: EGFP and streptavidin in solutions crowded with the 276.5 kDa dextran, and fluorescein and 282 kDa FITC-dextran in solutions crowded with 401.3 kDa
    Figure Legend Snippet: Anomalous diffusion exponent as a function of dextran concentration fitted to Eq. 10 for various tracers: EGFP and streptavidin in solutions crowded with the 276.5 kDa dextran, and fluorescein and 282 kDa FITC-dextran in solutions crowded with 401.3 kDa

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Anomalous diffusion exponents associated with the diffusion of streptavidin in solutions crowded with either BSA or nonfluorescent streptavidin for different concentrations of the obstacle proteins fitted to Eq. 10. For comparison, the exponent associated
    Figure Legend Snippet: Anomalous diffusion exponents associated with the diffusion of streptavidin in solutions crowded with either BSA or nonfluorescent streptavidin for different concentrations of the obstacle proteins fitted to Eq. 10. For comparison, the exponent associated

    Techniques Used: Diffusion-based Assay

    ( a ) Apparent diffusion coefficient, D ( τ D ), associated with the diffusion of streptavidin as a function of dextran concentration for dextrans of various average molecular weights ( open and solid symbols ). Also shown is D ( τ D ) for a 282 kDa
    Figure Legend Snippet: ( a ) Apparent diffusion coefficient, D ( τ D ), associated with the diffusion of streptavidin as a function of dextran concentration for dextrans of various average molecular weights ( open and solid symbols ). Also shown is D ( τ D ) for a 282 kDa

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Anomalous diffusion exponent α associated with the diffusion of streptavidin in presence of 75 g/l and 200 g/l of 276 kDa dextran as a function of the temperature. The average values of the anomalous diffusion exponent α over the considered
    Figure Legend Snippet: Anomalous diffusion exponent α associated with the diffusion of streptavidin in presence of 75 g/l and 200 g/l of 276 kDa dextran as a function of the temperature. The average values of the anomalous diffusion exponent α over the considered

    Techniques Used: Diffusion-based Assay

    Anomalous diffusion exponent corresponding to the diffusion of streptavidin in PBS with and without 200 g/l of 276 kDa dextran, as a function of added NaCl.
    Figure Legend Snippet: Anomalous diffusion exponent corresponding to the diffusion of streptavidin in PBS with and without 200 g/l of 276 kDa dextran, as a function of added NaCl.

    Techniques Used: Diffusion-based Assay

    16) Product Images from "Mutation Screening Based on the Mechanical Properties of DNA Molecules Tethered to a Solid Surface"

    Article Title: Mutation Screening Based on the Mechanical Properties of DNA Molecules Tethered to a Solid Surface

    Journal:

    doi: 10.1021/jp909501h

    Plot of raw ( blue curve ) and fitted ( black curve ) data for a typical frequency response and energy dissipation of the resonator at its fifth harmonic resonance as a function of time during the immobilization of the streptavidin layer and the ssDNA film.
    Figure Legend Snippet: Plot of raw ( blue curve ) and fitted ( black curve ) data for a typical frequency response and energy dissipation of the resonator at its fifth harmonic resonance as a function of time during the immobilization of the streptavidin layer and the ssDNA film.

    Techniques Used:

    Schematic process for immobilizing ssDNA strands with a complex 3-D structure on a quartz crystal. (a) A monolayer of biotin-thiol is immobilized onto the Au film by covalently linking the thiol group to Au. (b) A layer of streptavidin binds to the biotin
    Figure Legend Snippet: Schematic process for immobilizing ssDNA strands with a complex 3-D structure on a quartz crystal. (a) A monolayer of biotin-thiol is immobilized onto the Au film by covalently linking the thiol group to Au. (b) A layer of streptavidin binds to the biotin

    Techniques Used:

    17) Product Images from "Liposomes functionalized to overcome the blood-brain barrier and to target amyloid-? peptide: the chemical design affects the permeability across an in vitro model"

    Article Title: Liposomes functionalized to overcome the blood-brain barrier and to target amyloid-? peptide: the chemical design affects the permeability across an in vitro model

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S42783

    Binding of LIP to Aβ fibrils. SPR sensorgrams resulting from the binding of ( A ) PA-LIP or ( B ) b/s-RI-PA-LIP or ( C ) cov-RI-PA-LIP on Aβ fibrils covalently immobilized on the gold sensor surface. Note: LIP were injected at two concentrations of total lipids (100 μM and 300 μM), corresponding to 2.5 μM and 7.5 μM of exposed PA. Abbreviations: Aβ, Aβ1-42 peptide; b/s, biotin/streptavidin; cov, covalent; LIP, liposomes; PA, phosphatidic acid; RI, RI7217 antibody; SPR, surface plasmon resonance.
    Figure Legend Snippet: Binding of LIP to Aβ fibrils. SPR sensorgrams resulting from the binding of ( A ) PA-LIP or ( B ) b/s-RI-PA-LIP or ( C ) cov-RI-PA-LIP on Aβ fibrils covalently immobilized on the gold sensor surface. Note: LIP were injected at two concentrations of total lipids (100 μM and 300 μM), corresponding to 2.5 μM and 7.5 μM of exposed PA. Abbreviations: Aβ, Aβ1-42 peptide; b/s, biotin/streptavidin; cov, covalent; LIP, liposomes; PA, phosphatidic acid; RI, RI7217 antibody; SPR, surface plasmon resonance.

    Techniques Used: Binding Assay, SPR Assay, Injection

    Theoretical structure of RI-PA-LIP. RI7217 antibody was linked to LIP by ( A ) biotin/streptavidin ligation technique or ( B ) covalent coupling via thiol–maleimide reaction. Abbreviations: Chol, cholesterol; LIP, liposomes; mAb, monoclonal antibody; [ 14 C]-PA, 14C-radiolabelled phosphatidic acid; [ 3 H]-Sm, tritiated sphingomyelin; PA, phosphatidic acid; Sm, sphingomyelin.
    Figure Legend Snippet: Theoretical structure of RI-PA-LIP. RI7217 antibody was linked to LIP by ( A ) biotin/streptavidin ligation technique or ( B ) covalent coupling via thiol–maleimide reaction. Abbreviations: Chol, cholesterol; LIP, liposomes; mAb, monoclonal antibody; [ 14 C]-PA, 14C-radiolabelled phosphatidic acid; [ 3 H]-Sm, tritiated sphingomyelin; PA, phosphatidic acid; Sm, sphingomyelin.

    Techniques Used: Ligation

    CLSM of hCMEC/D3 cells after incubation with LIP. hCMEC/D3 cells were incubated with fluorescent labeled LIP (200 nmols of total lipids) for 2 hours at 37°C, 5% CO 2 saturation. hCMEC/D3 cells after incubation with ( A ) PA-LIP, ( B ) LIP b/s-RI-PA-LIP, or ( C ) cov-RI-PA-LIP. Notes: Cells were incubated with phalloidin in order to visualize the actin filaments (red fluorescence); nuclear staining was performed by DAPI (blue staining) and BODIPY-Sm to mark LIP (green staining). Scale bar = 20 μM. Abbreviations: b/s, biotin/streptavidin; BODIPY, boron-dipyrromethene; CLSM,; cov, covalent; DAPI, 4′,6-diamidino-2-phenylindole; LIP, liposomes; PA, phosphatidic acid; CLSM, confocal laser scanning microscopy; RI, anti-transferrin receptor RI7217 antibody; Sm, sphingomyelin.
    Figure Legend Snippet: CLSM of hCMEC/D3 cells after incubation with LIP. hCMEC/D3 cells were incubated with fluorescent labeled LIP (200 nmols of total lipids) for 2 hours at 37°C, 5% CO 2 saturation. hCMEC/D3 cells after incubation with ( A ) PA-LIP, ( B ) LIP b/s-RI-PA-LIP, or ( C ) cov-RI-PA-LIP. Notes: Cells were incubated with phalloidin in order to visualize the actin filaments (red fluorescence); nuclear staining was performed by DAPI (blue staining) and BODIPY-Sm to mark LIP (green staining). Scale bar = 20 μM. Abbreviations: b/s, biotin/streptavidin; BODIPY, boron-dipyrromethene; CLSM,; cov, covalent; DAPI, 4′,6-diamidino-2-phenylindole; LIP, liposomes; PA, phosphatidic acid; CLSM, confocal laser scanning microscopy; RI, anti-transferrin receptor RI7217 antibody; Sm, sphingomyelin.

    Techniques Used: Confocal Laser Scanning Microscopy, Incubation, Labeling, Fluorescence, Staining

    Binding of LIP to TfR. ( A ) Dot blots of 20 ng TfR spotted in a PVDF membrane, incubated with cov-RI-PA-LIP (lane 1), or b/s-RI-PA-LIP (lane 2), or biotinylated RI7217 (lane 3), or thiolated RI7217 (lane 4), or native RI7217 (lane 5), or control IgG (lane 6). Spots were detected with HRP-conjugated IgG anti-mouse, followed by ECL detection. SPR sensorgrams resulting from the binding of ( B ) b/s-R-IPA- LIP or ( C ) cov-RI-PA-LIP on TfR covalently immobilized on the gold sensor surface. Note: LIP were injected at concentrations of exposed RI7217 of 10 to 600 nM. Abbreviations: b/s, biotin/streptavidin; cov, covalent; ECL,; HRP, horseradish peroxidise; Ig, immunoglobulin; LIP, liposomes; PA, phosphatidic acid; PVDF, polyvinylidene fluoride; RI, RI7217 antibody; SPR, surface plasmon resonance; TfR, transferrin receptor.
    Figure Legend Snippet: Binding of LIP to TfR. ( A ) Dot blots of 20 ng TfR spotted in a PVDF membrane, incubated with cov-RI-PA-LIP (lane 1), or b/s-RI-PA-LIP (lane 2), or biotinylated RI7217 (lane 3), or thiolated RI7217 (lane 4), or native RI7217 (lane 5), or control IgG (lane 6). Spots were detected with HRP-conjugated IgG anti-mouse, followed by ECL detection. SPR sensorgrams resulting from the binding of ( B ) b/s-R-IPA- LIP or ( C ) cov-RI-PA-LIP on TfR covalently immobilized on the gold sensor surface. Note: LIP were injected at concentrations of exposed RI7217 of 10 to 600 nM. Abbreviations: b/s, biotin/streptavidin; cov, covalent; ECL,; HRP, horseradish peroxidise; Ig, immunoglobulin; LIP, liposomes; PA, phosphatidic acid; PVDF, polyvinylidene fluoride; RI, RI7217 antibody; SPR, surface plasmon resonance; TfR, transferrin receptor.

    Techniques Used: Binding Assay, Incubation, SPR Assay, Indirect Immunoperoxidase Assay, Injection

    18) Product Images from "Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection"

    Article Title: Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

    Journal:

    doi: 10.1016/j.jsb.2008.07.008

    (A) Molecular representation of the hydrated streptavidin tetramer. (B) Projection map (32×32 pixels with a pixel size of 0.182 nm); (C) Projection map was Gaussian-filtered with a half power at 1/1.3 nm −1 ; (D) Data-derived projection
    Figure Legend Snippet: (A) Molecular representation of the hydrated streptavidin tetramer. (B) Projection map (32×32 pixels with a pixel size of 0.182 nm); (C) Projection map was Gaussian-filtered with a half power at 1/1.3 nm −1 ; (D) Data-derived projection

    Techniques Used: Derivative Assay

    Liposome tethering kinetics. EM images of negatively stained 2D streptavidin crystal (spanning holes in the perforated carbon film) with tethered liposomes (POPC liposomes doped with biotin-DPPE) at low (A), intermediate (B) and high (C–D) magnification.
    Figure Legend Snippet: Liposome tethering kinetics. EM images of negatively stained 2D streptavidin crystal (spanning holes in the perforated carbon film) with tethered liposomes (POPC liposomes doped with biotin-DPPE) at low (A), intermediate (B) and high (C–D) magnification.

    Techniques Used: Electron Microscopy, Staining

    Protein concentration and crystal growth time effects on the crystal size. EM images show the negatively stained crystal grown from (A) 0.2 mg/ml (B) 0.1 mg/ml (C) 0.05 mg/ml of streptavidin solutions for 2 hours at room temperature. The black curves
    Figure Legend Snippet: Protein concentration and crystal growth time effects on the crystal size. EM images show the negatively stained crystal grown from (A) 0.2 mg/ml (B) 0.1 mg/ml (C) 0.05 mg/ml of streptavidin solutions for 2 hours at room temperature. The black curves

    Techniques Used: Protein Concentration, Electron Microscopy, Staining

    19) Product Images from "Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts"

    Article Title: Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20022112

    Activation of lipid raft-associated Fyn is Lck dependent. TCRCβ and CD4 were coaggregated on CD4 + lymph node cells derived from wild-type (WT) or LGF + Lck −/− (Tg). (a) Total phosphotyrosyl content of each sample was assessed by immunoblotting with phophotyrosine-specific mAb (top panel). The filter was stripped and successively probed with anti-Lck (middle panel) followed by anti-Fyn (bottom panel). (b) Fyn immunoprecipitates were probed with pY418 Src and developed with Protein A-HRP (top panel). The filter was stripped and probed with anti-Fyn (middle panel). Quantification of pY417 Fyn normalized to total Fyn (bottom panel). (c) Distribution of GM1, Lck and Fyn in sucrose EDGC fractions derived from Tg cells subjected or not to streptavidin-mediated aggregation for 90 s. (d) Fyn immunoprecipitates derived from R and S fractions of WT and Tg cells were subjected to immune complex kinase assays. The phospho-enolase signals (pY Enolase) for Fyn (top panel) were normalized to total Fyn content (middle panel) and specific kinase activity was expressed as a ratio between pY Enolase and total Fyn (bottom panel). Bars representing R and S fractions from the same sample are grouped. Levels of pY417 Fyn as well as Fyn specific kinase activity in nonaggregated control samples were given a reference value “1.” Sample lanes in panels a and b are aligned over a common figure legend.
    Figure Legend Snippet: Activation of lipid raft-associated Fyn is Lck dependent. TCRCβ and CD4 were coaggregated on CD4 + lymph node cells derived from wild-type (WT) or LGF + Lck −/− (Tg). (a) Total phosphotyrosyl content of each sample was assessed by immunoblotting with phophotyrosine-specific mAb (top panel). The filter was stripped and successively probed with anti-Lck (middle panel) followed by anti-Fyn (bottom panel). (b) Fyn immunoprecipitates were probed with pY418 Src and developed with Protein A-HRP (top panel). The filter was stripped and probed with anti-Fyn (middle panel). Quantification of pY417 Fyn normalized to total Fyn (bottom panel). (c) Distribution of GM1, Lck and Fyn in sucrose EDGC fractions derived from Tg cells subjected or not to streptavidin-mediated aggregation for 90 s. (d) Fyn immunoprecipitates derived from R and S fractions of WT and Tg cells were subjected to immune complex kinase assays. The phospho-enolase signals (pY Enolase) for Fyn (top panel) were normalized to total Fyn content (middle panel) and specific kinase activity was expressed as a ratio between pY Enolase and total Fyn (bottom panel). Bars representing R and S fractions from the same sample are grouped. Levels of pY417 Fyn as well as Fyn specific kinase activity in nonaggregated control samples were given a reference value “1.” Sample lanes in panels a and b are aligned over a common figure legend.

    Techniques Used: Activation Assay, Derivative Assay, Immune Complex Kinase Assay, Activity Assay

    Coaggregation of TCR and CD4 induces the translocation of Lck into lipid rafts. (a) 10 7 primary CD4 + lymph node T cells were precoated with biotinylated anti-TCRCβ (H57 b ) and/or biotinylated anti-CD4 (GK1.5 b ) followed by the addition of Streptavidin for 10, 30, or 90 s, as indicated. Distribution of GM1, Lck, and Fyn was assessed in fractions 1–2 and 9–10, representing lipid raft (R) and soluble fractions (S), respectively. (b) Quantification of Lck subcellular distribution. Numbers represent the proportion of total Lck detected in R and S fractions. (c) Quantification of Lck enrichment in lipid rafts. The values represent the mean of three independent experiments performed as described for panel a with one SD indicated. (d) 10 7 primary CD4 + lymph node T cells were precoated with the indicated concentrations of H57 b and GK1.5 b and coaggregated or not by the addition of Streptavidin for 30 s. Distribution of Lck was assessed as described above. Quantification of the signals revealed was performed by densitometric analysis.
    Figure Legend Snippet: Coaggregation of TCR and CD4 induces the translocation of Lck into lipid rafts. (a) 10 7 primary CD4 + lymph node T cells were precoated with biotinylated anti-TCRCβ (H57 b ) and/or biotinylated anti-CD4 (GK1.5 b ) followed by the addition of Streptavidin for 10, 30, or 90 s, as indicated. Distribution of GM1, Lck, and Fyn was assessed in fractions 1–2 and 9–10, representing lipid raft (R) and soluble fractions (S), respectively. (b) Quantification of Lck subcellular distribution. Numbers represent the proportion of total Lck detected in R and S fractions. (c) Quantification of Lck enrichment in lipid rafts. The values represent the mean of three independent experiments performed as described for panel a with one SD indicated. (d) 10 7 primary CD4 + lymph node T cells were precoated with the indicated concentrations of H57 b and GK1.5 b and coaggregated or not by the addition of Streptavidin for 30 s. Distribution of Lck was assessed as described above. Quantification of the signals revealed was performed by densitometric analysis.

    Techniques Used: Translocation Assay

    Coaggregation of TCR and CD4 results in sequential activation of Lck then Fyn. (a) Total phosphotyrosyl content of each sample precoated with 1 μg/ml of biotinylated anti-TCRCβ (H57 b ( 1 )) and/or 0.3 μg/ml of biotinylated anti-CD4 (GK1.5 b (0.3)) and coaggregated with 50 μg/ml of streptavidin for 10, 30, and 90 s, was assessed by immuno-blotting with phosphotyrosine specific mAb. The filter was stripped and successively probed with anti-Lck followed by anti-Fyn. (b) Cell lysates probed with pY394 Lck. The filter was stripped and probed with anti-Lck. (c) Fyn immunoprecipitates were probed with pY418 Src. The filter was stripped and probed with anti-Fyn. Histograms in b and c show the quantification of pY394 Lck and pY417 Fyn normalized to total kinase signals. The nonaggregated control sample was given a reference value “1.” All sample lanes in a, b, and c are aligned over a common legend.
    Figure Legend Snippet: Coaggregation of TCR and CD4 results in sequential activation of Lck then Fyn. (a) Total phosphotyrosyl content of each sample precoated with 1 μg/ml of biotinylated anti-TCRCβ (H57 b ( 1 )) and/or 0.3 μg/ml of biotinylated anti-CD4 (GK1.5 b (0.3)) and coaggregated with 50 μg/ml of streptavidin for 10, 30, and 90 s, was assessed by immuno-blotting with phosphotyrosine specific mAb. The filter was stripped and successively probed with anti-Lck followed by anti-Fyn. (b) Cell lysates probed with pY394 Lck. The filter was stripped and probed with anti-Lck. (c) Fyn immunoprecipitates were probed with pY418 Src. The filter was stripped and probed with anti-Fyn. Histograms in b and c show the quantification of pY394 Lck and pY417 Fyn normalized to total kinase signals. The nonaggregated control sample was given a reference value “1.” All sample lanes in a, b, and c are aligned over a common legend.

    Techniques Used: Activation Assay

    20) Product Images from "Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma"

    Article Title: Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086642

    Video intensity from SFRP2 -targeted microbubble contrast agent correlated significantly with SVR angiosarcoma tumor volume. The baseline-subtracted average pixel intensity for each tumor was plotted against tumor volumes determined using our three-dimensional B-mode scans. Only 1 of 13 animals was observed to have higher signal for the streptavidin control than for the SFRP2 -targeted microbubbles (indicated by arrow). Correlation analysis showed a significant positive relationship between SFRP2 video signal and tumor volume as both increased together whether analyzed with (p = 0.048, Pearson r = 0.56) or without (p = 0.003, Pearson r = 0.78) the single “outlier” animal. Linear regression was used to determine the best-fit line through all data points to represent this relationship.
    Figure Legend Snippet: Video intensity from SFRP2 -targeted microbubble contrast agent correlated significantly with SVR angiosarcoma tumor volume. The baseline-subtracted average pixel intensity for each tumor was plotted against tumor volumes determined using our three-dimensional B-mode scans. Only 1 of 13 animals was observed to have higher signal for the streptavidin control than for the SFRP2 -targeted microbubbles (indicated by arrow). Correlation analysis showed a significant positive relationship between SFRP2 video signal and tumor volume as both increased together whether analyzed with (p = 0.048, Pearson r = 0.56) or without (p = 0.003, Pearson r = 0.78) the single “outlier” animal. Linear regression was used to determine the best-fit line through all data points to represent this relationship.

    Techniques Used:

    Modified microbubble contrast agents were not retained at significant levels in nonmalignant vasculature. B-mode images (black and white) are shown overlaid with CPS-mode images (green). CPS-mode is sensitive to ultrasound signal typically produced by microbubbles oscillating within an ultrasound field. (A) In the absence of ultrasound contrast agent (no contrast) there was no CPS-mode signal within the region of interest (dotted rectangle) outside of the tumor margins. Tissue artifacts generated the CPS-mode signal observed in the absence of contrast agent. Contrast agent freely flowing through both tumor and non-tumor vasculature generated CPS-mode signal throughout the field of view (panel A, perfusion, middle frames) with either streptavidin-coated (control, upper frames) or SFRP2- targeted ultrasound contrast agent (lower frames). No signal remained within the region of interest drawn outside of the tumor margins after allowing all freely flowing contrast agent to be cleared from the vasculature, while SFRP2 specific signal was retained within the tumor margins. (B) Modified microbubble contrast agents were not retained within kidney vasculature. Freely flowing streptavidin-loaded microbubbles (panel B, control, upper frames) or SFRP2 - targeted microbubbles (panel B, lower frames) were allowed to clear from the vasculature prior to three-dimensional molecular imaging. Single frames are shown from two different animals (animal 1 or animal 2). The dotted oval region of interest represents the location of the kidney (K) and there was no significant difference in average pixel intensity after injection of either streptavidin-loaded or SFRP2 -targeted microbubbles. In contrast, the liver (L) retained both modified microbubble contrast agents to a high degree. White scale bars in panels A and B represent 5 mm.
    Figure Legend Snippet: Modified microbubble contrast agents were not retained at significant levels in nonmalignant vasculature. B-mode images (black and white) are shown overlaid with CPS-mode images (green). CPS-mode is sensitive to ultrasound signal typically produced by microbubbles oscillating within an ultrasound field. (A) In the absence of ultrasound contrast agent (no contrast) there was no CPS-mode signal within the region of interest (dotted rectangle) outside of the tumor margins. Tissue artifacts generated the CPS-mode signal observed in the absence of contrast agent. Contrast agent freely flowing through both tumor and non-tumor vasculature generated CPS-mode signal throughout the field of view (panel A, perfusion, middle frames) with either streptavidin-coated (control, upper frames) or SFRP2- targeted ultrasound contrast agent (lower frames). No signal remained within the region of interest drawn outside of the tumor margins after allowing all freely flowing contrast agent to be cleared from the vasculature, while SFRP2 specific signal was retained within the tumor margins. (B) Modified microbubble contrast agents were not retained within kidney vasculature. Freely flowing streptavidin-loaded microbubbles (panel B, control, upper frames) or SFRP2 - targeted microbubbles (panel B, lower frames) were allowed to clear from the vasculature prior to three-dimensional molecular imaging. Single frames are shown from two different animals (animal 1 or animal 2). The dotted oval region of interest represents the location of the kidney (K) and there was no significant difference in average pixel intensity after injection of either streptavidin-loaded or SFRP2 -targeted microbubbles. In contrast, the liver (L) retained both modified microbubble contrast agents to a high degree. White scale bars in panels A and B represent 5 mm.

    Techniques Used: Modification, Produced, Generated, Imaging, Injection

    Size distribution of ultrasound microbubble contrast agent bound to SFRP2 antibody via a streptavidin bridge. Microbubble contrast agent containing biotinylated lipid was size-sorted by differential centrifugation, prior to sequential incubations with streptavidin and the SFRP2 antibody mixture. Aliquots of the contrast agent were used to determine the microbubble concentration and size distribution ( Table 1 ).
    Figure Legend Snippet: Size distribution of ultrasound microbubble contrast agent bound to SFRP2 antibody via a streptavidin bridge. Microbubble contrast agent containing biotinylated lipid was size-sorted by differential centrifugation, prior to sequential incubations with streptavidin and the SFRP2 antibody mixture. Aliquots of the contrast agent were used to determine the microbubble concentration and size distribution ( Table 1 ).

    Techniques Used: Centrifugation, Concentration Assay

    SFRP2 -targeted microbubbles bound specifically to vasculature within angiosarcoma. B-mode images of the SVR angiosarcoma tumors were overlaid in green with molecular images of (A) control streptavidin loaded microbubbles or (B) SFRP2 -targeted microbubbles after three-dimensional molecular imaging. The average pixel intensity observed for SFRP2 -targeted imaging was significantly higher (*p = 0.003, n = 13, paired t-test, two-tailed) than observed for the streptavidin control (C). Immunohistochemistry demonstrated high levels of expression for SFRP2 in angiosarcoma (D). Black scale bars in panels A and B represent 1 mm. Black scale bars in panels D and E represent 35 µm.
    Figure Legend Snippet: SFRP2 -targeted microbubbles bound specifically to vasculature within angiosarcoma. B-mode images of the SVR angiosarcoma tumors were overlaid in green with molecular images of (A) control streptavidin loaded microbubbles or (B) SFRP2 -targeted microbubbles after three-dimensional molecular imaging. The average pixel intensity observed for SFRP2 -targeted imaging was significantly higher (*p = 0.003, n = 13, paired t-test, two-tailed) than observed for the streptavidin control (C). Immunohistochemistry demonstrated high levels of expression for SFRP2 in angiosarcoma (D). Black scale bars in panels A and B represent 1 mm. Black scale bars in panels D and E represent 35 µm.

    Techniques Used: Imaging, Two Tailed Test, Immunohistochemistry, Expressing

    Microbubbles targeted with anti-chicken IgY were retained within angiosarcoma vasculature at significantly lower levels than microbubbles loaded with streptavidin. Streptavidin-coated microbubbles were bound to a mixture of biotinylated anti-chicken IgY (raised in rabbit and goat) to produce anti-chicken IgY control microbubbles. Three-dimensional molecular imaging with the anti-chicken IgY control microbubbles resulted in significantly lower average pixel intensity (*p = 0.0002, n = 10, unpaired t-test, two-tailed) than observed with microbubbles coated only with streptavidin.
    Figure Legend Snippet: Microbubbles targeted with anti-chicken IgY were retained within angiosarcoma vasculature at significantly lower levels than microbubbles loaded with streptavidin. Streptavidin-coated microbubbles were bound to a mixture of biotinylated anti-chicken IgY (raised in rabbit and goat) to produce anti-chicken IgY control microbubbles. Three-dimensional molecular imaging with the anti-chicken IgY control microbubbles resulted in significantly lower average pixel intensity (*p = 0.0002, n = 10, unpaired t-test, two-tailed) than observed with microbubbles coated only with streptavidin.

    Techniques Used: Imaging, Two Tailed Test

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    Article Snippet: Polystyrene microtiter plates (Maxisorp by NUNC, Naperville, IL, USA) were coated with monoclonal anti-LL-37 (5 μg/ml) [ ] in carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate and 0.02% sodium azide [pH 9.6]) and incubated overnight at 4°C. .. After sequential incubations with biotinylated rabbit anti-LL-37 (1 μg/mL) (Innovagen) and Streptavidin-alkaline phosphatase conjugate (Chemicon, Melbourne, Australia) for 2h each at RT, the reaction was developed using 4-methyl-umbelliferyl phosphate as the substrate (Molecular Probes, Leiden, The Netherlands).

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Human salivary α-amylase type IX-A, control mucin from bovine submaxillary glands (type I-S), and human salivary α-amylase-specific rabbit antibody were purchased from Sigma. .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization. .. A DNA fragment containing gtfB from S. mutans was removed from plasmid pYNB13 ( ) (provided by H. K. Kuramitsu, State University of New York at Buffalo, Buffalo, N.Y.) by digestion with Xho I and Not I and was subcloned into expression vector pET32b(+) (Novagen, Madison, Wis.).

    Article Title:
    Article Snippet: For assays with immobilized Cstn3, 200 ngr Cstn3 (or fragments) in binding buffer/Ca2+ was coated for 1 h at 150 rpm, and the wells were treated as described above except that they were blocked with blocking buffer containing 3% gelatin. .. Wells were then incubated with increasing concentrations of biotinylated neurexin 1α* in binding buffer/Ca2+ (in triplicate) for 1 h. To develop the signal, wells were incubated with anti-streptavidin HRP conjugate (Sigma S2438; diluted 1:5000 in blocking buffer) for 45 min, washed three times with binding buffer/Ca2+ or binding buffer/EDTA, and then incubated with the substrate o -phenylenediamine (Calbiochem) for 10 min. .. The reaction was stopped by adding 50 μl/well 0.5 m H2 SO4 , and the absorbance was read at 490 nm.

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: After ultracentrifugation at 180,000 × g for 15 min, the supernatant was diluted 10-fold in PA3-8 buffer and incubated with preequilibrated Ni-NTA resin for 60 min. .. The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich).

    Article Title: CD84 negatively regulates IgE high affinity receptor signaling in human mast cells
    Article Snippet: Cells were then incubated with biotinylated anti-CD84 or anti-CD229 mAbs as isotype control in Tyrode´s buffer (10 mM Hepes, 137 mM NaCl, 2.7 mM, KCl, 0.4 mM NaH2 PO4 , 1.8 mM CaCl2 , 1.3 mM MgSO4 , 5.6 mM glucose and 0.025% BSA) for 30 min at 37 °C. .. After washing, cells were stimulated with 100 ng/ml streptavidin (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. Several streptavidin concentrations were tested in order to verify excess of streptavidin in the assay and lack of competition between biotinylated IgE and either biotinylated anti-CD84 or isotype control.

    Article Title: Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding
    Article Snippet: A standard curve was included on each plate with purified c-Myc HSA diluted in 10% cynomologus monkey plasma in PBST. .. Next 100 μl of an biotinylated anti-HSA antibody (Abcam) at 1.5 μg/ml in PBST was added to the plates followed incubation for 30 min. Plates were washed as above, and 100 μl of 1.25 μg/ml HRP-conjugated streptavidin (Sigma-Aldrich) in PBST was added to the plates and incubated for 30 min. .. Subsequently, the plates were washed as above, and the signal was developed with 100 μl of 3,3′,5,5′-tetramethylbenzidine-Ultra substrate (Pierce) for 5 min before the reactions were stopped with 0.2 m sulfuric acid.

    Activity Assay:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore). .. Immunoreactive products were visualized by chemiluminescence using the ECL Plus kit (Amersham Biosciences), and their relative intensity was evaluated with the aid of Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).

    Article Title: Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1? (MIP-1?)-dependent Pathways
    Article Snippet: Levamisole (Vector Laboratories, Inc.), at a final concentration of 200 μM, was used to inhibit endogenous alkaline phosphatase activity. .. Equivalent concentrations of rabbit, rat, goat or mouse IgG were used as control antibodies ( Sigma Chemical Co. ).

    Article Title: CD84 negatively regulates IgE high affinity receptor signaling in human mast cells
    Article Snippet: After washing, cells were stimulated with 100 ng/ml streptavidin (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. Several streptavidin concentrations were tested in order to verify excess of streptavidin in the assay and lack of competition between biotinylated IgE and either biotinylated anti-CD84 or isotype control. .. After washing, cells were stimulated with 100 ng/ml streptavidin (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. Several streptavidin concentrations were tested in order to verify excess of streptavidin in the assay and lack of competition between biotinylated IgE and either biotinylated anti-CD84 or isotype control.

    Expressing:

    Article Title: Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial
    Article Snippet: After sequential incubations with biotinylated rabbit anti-LL-37 (1 μg/mL) (Innovagen) and Streptavidin-alkaline phosphatase conjugate (Chemicon, Melbourne, Australia) for 2h each at RT, the reaction was developed using 4-methyl-umbelliferyl phosphate as the substrate (Molecular Probes, Leiden, The Netherlands). .. Fluorescence was measured at an excitation wavelength of 360 nm and emission wavelength of 450 nm. mRNA was extracted from MDM and NAL using the RNeasy Mini kit as described by the manufacturer (Qiagen GmbH). mRNA was reverse-transcribed using Bio-Rad CFX 1000, (Hercules, CA, USA) and cDNA was synthesized using Superscript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA).

    BIA-KA:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Cell and tissue extracts were prepared and assayed for total protein content using the BCA kit (Pierce) ( ). .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Article Title: Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment
    Article Snippet: Anti-ubiquitin antibody (U5379), 6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS102, N179), (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK801, M107), 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466, G119), GSNO (N4148), dl -DTT (43817), Z-Leu-Leu-Leu-al (MG132, C2211), N -acetyl- l -cysteine (NAC, A7250), neocuproine (N1501), methyl methylthiomethyl sulfoxide (MMTS, 177954), (+)-sodium l -ascorbate (A7631), streptavidin-agarose (A1638), and Z-Leu-Leu-Glu β-naphthylamide (Z-Leu-Leu-Glu βNA, C0788) were purchased from Sigma-Aldrich (Poole, U.K.). .. Guava TUNEL kit (4500–0121) was purchased from Millipore Co. (Bedford, MA).

    Modification:

    Article Title: The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses
    Article Snippet: Cells were irradiated and incubated with 5 μM Fluo-3/AM in modified Tyrode’s buffer (without 1 mM CaCl2 ) for 30 min at 37°C. .. After washing, cells were stimulated with 1 μg/ml anti-IgE (Dako) and 0.01 μg/ml streptavidin (Sigma).

    Western Blot:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Immunoblots, Immunohistochemistry, and in Vitro Kinase Assay —VSMC were cultured for 4 days in 10% FBS and for 2 additional days in 2% FBS. .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Article Title: Serological Markers of Sand Fly Exposure to Evaluate Insecticidal Nets against Visceral Leishmaniasis in India and Nepal: A Cluster-Randomized Trial
    Article Snippet: Plates were incubated with biotinylated goat anti-human IgG (1∶1000 in PBS-T, Sigma) for 1 hr at 25°C, washed and incubated with streptavidin-conjugated alkaline phosphatase (1∶1000 dilution in PBS-T, Sigma) for a further 1 hr at 25°C. .. To minimise day to day variation in ELISA performance three sera from the same individual collected over the entire trial (baseline; 12 and 24 months follow-up samples) were processed in the same plate.

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Human salivary α-amylase type IX-A, control mucin from bovine submaxillary glands (type I-S), and human salivary α-amylase-specific rabbit antibody were purchased from Sigma. .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization. .. A DNA fragment containing gtfB from S. mutans was removed from plasmid pYNB13 ( ) (provided by H. K. Kuramitsu, State University of New York at Buffalo, Buffalo, N.Y.) by digestion with Xho I and Not I and was subcloned into expression vector pET32b(+) (Novagen, Madison, Wis.).

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: The Ni-NTA resin was washed with 10 column volumes of W2 buffer and eluted with 3 column volumes of E2 buffer. .. The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich). .. For MPB labeling of proteoliposomes containing MrpG subunits with an introduced cysteine in the C-terminal segment, the proteoliposomes (300 μl) were mixed with 0.1 mM MPB and dispensed as two aliquots (150 μl each).

    Kinase Assay:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Immunoblots, Immunohistochemistry, and in Vitro Kinase Assay —VSMC were cultured for 4 days in 10% FBS and for 2 additional days in 2% FBS. .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    TUNEL Assay:

    Article Title: Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment
    Article Snippet: Anti-ubiquitin antibody (U5379), 6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS102, N179), (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK801, M107), 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466, G119), GSNO (N4148), dl -DTT (43817), Z-Leu-Leu-Leu-al (MG132, C2211), N -acetyl- l -cysteine (NAC, A7250), neocuproine (N1501), methyl methylthiomethyl sulfoxide (MMTS, 177954), (+)-sodium l -ascorbate (A7631), streptavidin-agarose (A1638), and Z-Leu-Leu-Glu β-naphthylamide (Z-Leu-Leu-Glu βNA, C0788) were purchased from Sigma-Aldrich (Poole, U.K.). .. Anti-ubiquitin antibody (U5379), 6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS102, N179), (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK801, M107), 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466, G119), GSNO (N4148), dl -DTT (43817), Z-Leu-Leu-Leu-al (MG132, C2211), N -acetyl- l -cysteine (NAC, A7250), neocuproine (N1501), methyl methylthiomethyl sulfoxide (MMTS, 177954), (+)-sodium l -ascorbate (A7631), streptavidin-agarose (A1638), and Z-Leu-Leu-Glu β-naphthylamide (Z-Leu-Leu-Glu βNA, C0788) were purchased from Sigma-Aldrich (Poole, U.K.).

    Immunohistochemistry:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: Paragraph title: Histology — In Situ Hybridization and Immunohistochemistry ... The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time.

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Immunoblots, Immunohistochemistry, and in Vitro Kinase Assay —VSMC were cultured for 4 days in 10% FBS and for 2 additional days in 2% FBS. .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Article Title: Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1? (MIP-1?)-dependent Pathways
    Article Snippet: Paragraph title: Immunohistochemistry. ... Equivalent concentrations of rabbit, rat, goat or mouse IgG were used as control antibodies ( Sigma Chemical Co. ).

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: For immunohistochemical analysis, cells in 3D gel were fixed with 10% neutral formalin for 10 minutes. .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Article Title: c-Jun-dependent β3GnT8 promotes tumorigenesis and metastasis of hepatocellular carcinoma by inducing CD147 glycosylation and altering N-glycan patterns
    Article Snippet: Paragraph title: Immunohistochemistry (IHC) staining and tissue microarray analysis ... Tissue microarray slides containing 75 pairs of liver cancer tissue and adjacent paracancer tissue arrays were purchased from Outdo Biotech (Shanghai, China) and stained with a rabbit anti-β3GnT8 antibody and HRP-conjugated anti-rabbit IgG secondary antibody (Beyotime, Haimen, China) or biotinylated-lycppersicon esculentum (Tomato) lectin (LEL) and HRP-conjugated streptavidin (Sigama, Sigma, St. Louis, MO, USA).

    Immunoprecipitation:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore). .. Immunoreactive products were visualized by chemiluminescence using the ECL Plus kit (Amersham Biosciences), and their relative intensity was evaluated with the aid of Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).

    Cell Culture:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Immunoblots, Immunohistochemistry, and in Vitro Kinase Assay —VSMC were cultured for 4 days in 10% FBS and for 2 additional days in 2% FBS. .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Hemagglutination Assay:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: CD8-enriched T cells ( > 90% CD8+ by flow cytometry) were then plated in triplicate for 24 h on Millipore HA membrane 96-well plates coated with anti-gamma interferon antibody (clone MD-1; BioSource International, Inc., Camarillo, Calif.). .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Light Microscopy:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: The center-to-edge images were recorded using a light microscope by adjusting the focus plain on the z-stack. .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Generated:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time. .. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was done according to the manufacturer’s protocol (Zymed) to measure cell proliferation.

    Article Title: Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial
    Article Snippet: A standard curve of the ELISA was generated from synthetic LL-37 (Innovagen, Lund, Sweden). .. After sequential incubations with biotinylated rabbit anti-LL-37 (1 μg/mL) (Innovagen) and Streptavidin-alkaline phosphatase conjugate (Chemicon, Melbourne, Australia) for 2h each at RT, the reaction was developed using 4-methyl-umbelliferyl phosphate as the substrate (Molecular Probes, Leiden, The Netherlands).

    other:

    Article Title: Discovery of a Novel Series of Inhibitors of Lymphoid Tyrosine Phosphatase with Activity in Human T Cells
    Article Snippet: Streptavidin was obtained from Sigma and the F(ab′)2 cross-linker was from Jackson Immunolabs (West Grove, PA).

    Imaging:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL). .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Polymerase Chain Reaction:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time. .. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was done according to the manufacturer’s protocol (Zymed) to measure cell proliferation.

    Sonication:

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: To prepare samples with complete reaction between MPB and cysteine residues, the DM-solubilized membrane vesicles were sonicated briefly before adding 20 mM 2-mercaptoethanol to stop the labeling reaction. .. The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich).

    Quantitative RT-PCR:

    Article Title: Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial
    Article Snippet: After sequential incubations with biotinylated rabbit anti-LL-37 (1 μg/mL) (Innovagen) and Streptavidin-alkaline phosphatase conjugate (Chemicon, Melbourne, Australia) for 2h each at RT, the reaction was developed using 4-methyl-umbelliferyl phosphate as the substrate (Molecular Probes, Leiden, The Netherlands). .. Fluorescence was measured at an excitation wavelength of 360 nm and emission wavelength of 450 nm. mRNA was extracted from MDM and NAL using the RNeasy Mini kit as described by the manufacturer (Qiagen GmbH). mRNA was reverse-transcribed using Bio-Rad CFX 1000, (Hercules, CA, USA) and cDNA was synthesized using Superscript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA).

    Recombinant:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore). .. To assay p38 MAPK activity in vitro , cell lysates from wild-type and mutant VSMC cultures were immunoprecipitated overnight at 4 °C with an immobilized anti-phospho-p38 MAPK monoclonal antibody (Thr-180/Tyr-182; Cell Signaling Technology).

    Immunofluorescence:

    Article Title: Modulation of T cell proliferation and cytokine response by Plumbagin, extracted from Plumbago zeylanica in collagen induced arthritis
    Article Snippet: The experimental protocol was approved by the Institutional ethics committee, Andhra University, Visakhapatnam, Andhra Pradesh, India. .. Bovine Type II collagen, Freund's Complete Adjuvant (FCA), Incomplete Freund's Adjuvant (IFA), Goat anti-mouse IgG, Bovine Serum Albumin (BSA), O-phenylenediamine (OPD), Hydrogen peroxide, RPMI-1640, Hank's balanced salt solution (HBSS), Fetal Calf Serum (FCS), Ammonium Chloride and Streptavidin purchased from Sigma Chemical Company (St. Louis, MO, USA). .. Tritiated thymidine was supplied by Bhabha Atomic Research Centre (BARC), Mumbai, India.

    Nucleic Acid Electrophoresis:

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed by using the Mini-PROTEAN 3 and Mini Trans-Blot cell systems, respectively, as suggested by the manufacturer (Bio-Rad, Hercules, Calif.). .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization.

    Fluorescence:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL). .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Article Title: The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses
    Article Snippet: Cytosolic calcium levels were measured using the calcium-reactive fluorescence probe Fluo-3/AM (Molecular Probes, Eugene, OR). .. After washing, cells were stimulated with 1 μg/ml anti-IgE (Dako) and 0.01 μg/ml streptavidin (Sigma).

    Magnetic Beads:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: Following this culture period, nonadherent cells enriched for CD8+ cells were obtained using negative selection with anti-CD4 magnetic beads (Miltenyi Biotec, Auburn, Calif.). .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Mutagenesis:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore). .. Immunoreactive products were visualized by chemiluminescence using the ECL Plus kit (Amersham Biosciences), and their relative intensity was evaluated with the aid of Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).

    Isolation:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: To measure the frequency of in vitro-activatable SIV-specific CD8+ T cells, mononuclear cells were isolated by Ficoll-Hypaque density centrifugation. .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Flow Cytometry:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: CD8-enriched T cells ( > 90% CD8+ by flow cytometry) were then plated in triplicate for 24 h on Millipore HA membrane 96-well plates coated with anti-gamma interferon antibody (clone MD-1; BioSource International, Inc., Camarillo, Calif.). .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Microscopy:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL). .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Purification:

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Human salivary α-amylase type IX-A, control mucin from bovine submaxillary glands (type I-S), and human salivary α-amylase-specific rabbit antibody were purchased from Sigma. .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization. .. A DNA fragment containing gtfB from S. mutans was removed from plasmid pYNB13 ( ) (provided by H. K. Kuramitsu, State University of New York at Buffalo, Buffalo, N.Y.) by digestion with Xho I and Not I and was subcloned into expression vector pET32b(+) (Novagen, Madison, Wis.).

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: The cells were stimulated with 3 μg (SIV p28CA equivalent) of purified aldrithiol-2-inactivated SIV virions per ml in complete medium for 4 days. .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Article Title: Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding
    Article Snippet: A standard curve was included on each plate with purified c-Myc HSA diluted in 10% cynomologus monkey plasma in PBST. .. Next 100 μl of an biotinylated anti-HSA antibody (Abcam) at 1.5 μg/ml in PBST was added to the plates followed incubation for 30 min. Plates were washed as above, and 100 μl of 1.25 μg/ml HRP-conjugated streptavidin (Sigma-Aldrich) in PBST was added to the plates and incubated for 30 min.

    Sequencing:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time. .. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was done according to the manufacturer’s protocol (Zymed) to measure cell proliferation.

    Immunostaining:

    Article Title: c-Jun-dependent β3GnT8 promotes tumorigenesis and metastasis of hepatocellular carcinoma by inducing CD147 glycosylation and altering N-glycan patterns
    Article Snippet: Tissue microarray slides containing 75 pairs of liver cancer tissue and adjacent paracancer tissue arrays were purchased from Outdo Biotech (Shanghai, China) and stained with a rabbit anti-β3GnT8 antibody and HRP-conjugated anti-rabbit IgG secondary antibody (Beyotime, Haimen, China) or biotinylated-lycppersicon esculentum (Tomato) lectin (LEL) and HRP-conjugated streptavidin (Sigama, Sigma, St. Louis, MO, USA). .. The slides were blindly evaluated by an independent pathologist to determine the percentage of positive cells and the staining intensity.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: Imaging was taken by EVOS fluorescence microscope (Advanced Microscopy Group, WA) directly in culture wells. .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL). .. MSC and SCC-71 were labeled with Mitotracker green and red (Cat.M7512, Lifetech, CA) respectively before co-culture.

    Silver Staining:

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Gels were stained with either Bio-Safe Coomassie blue (Bio-Rad), silver stain (Bio-Rad), or Schiff's reagent (Sigma) as recommended by the manufacturers. .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization.

    In Situ Hybridization:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: Paragraph title: Histology — In Situ Hybridization and Immunohistochemistry ... The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time.

    Plasmid Preparation:

    Article Title: Stress Fracture Healing: Fatigue Loading of the Rat Ulna Induces Upregulation in Expression of Osteogenic and Angiogenic Genes that Mimic the Intramembranous Portion of Fracture Repair
    Article Snippet: The next day, slides were washed, incubated with a 1:500 dilution of Streptavidin (Sigma), washed, incubated with 1:200 of secondary antibody, washed, then visualized using 3,3′-diaminobenzidine (DAB) for a predetermined time. .. For in situ hybridization, the BSP sequence generated as a PCR standard was used as a probe [ ].

    Double Immunostaining:

    Article Title: Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1? (MIP-1?)-dependent Pathways
    Article Snippet: To detect MCMV antigen in double-immunostaining experiments, slides were blocked in 5% normal fetal bovine serum before overnight incubation at 4°C with mouse anti-MCMV immune serum. .. Equivalent concentrations of rabbit, rat, goat or mouse IgG were used as control antibodies ( Sigma Chemical Co. ).

    Real-time Polymerase Chain Reaction:

    Article Title: Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial
    Article Snippet: After sequential incubations with biotinylated rabbit anti-LL-37 (1 μg/mL) (Innovagen) and Streptavidin-alkaline phosphatase conjugate (Chemicon, Melbourne, Australia) for 2h each at RT, the reaction was developed using 4-methyl-umbelliferyl phosphate as the substrate (Molecular Probes, Leiden, The Netherlands). .. Fluorescence was measured at an excitation wavelength of 360 nm and emission wavelength of 450 nm. mRNA was extracted from MDM and NAL using the RNeasy Mini kit as described by the manufacturer (Qiagen GmbH). mRNA was reverse-transcribed using Bio-Rad CFX 1000, (Hercules, CA, USA) and cDNA was synthesized using Superscript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA).

    Selection:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: Following this culture period, nonadherent cells enriched for CD8+ cells were obtained using negative selection with anti-CD4 magnetic beads (Miltenyi Biotec, Auburn, Calif.). .. The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    In Vitro:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Immunoblots, Immunohistochemistry, and in Vitro Kinase Assay —VSMC were cultured for 4 days in 10% FBS and for 2 additional days in 2% FBS. .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Labeling:

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: Paragraph title: Sidedness assay of C terminus of MrpG by cysteine labeling. ... The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich).

    Irradiation:

    Article Title: The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses
    Article Snippet: Cells were irradiated and incubated with 5 μM Fluo-3/AM in modified Tyrode’s buffer (without 1 mM CaCl2 ) for 30 min at 37°C. .. After washing, cells were stimulated with 1 μg/ml anti-IgE (Dako) and 0.01 μg/ml streptavidin (Sigma).

    Concentration Assay:

    Article Title: Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1? (MIP-1?)-dependent Pathways
    Article Snippet: Levamisole (Vector Laboratories, Inc.), at a final concentration of 200 μM, was used to inhibit endogenous alkaline phosphatase activity. .. Equivalent concentrations of rabbit, rat, goat or mouse IgG were used as control antibodies ( Sigma Chemical Co. ).

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich). .. For MPB labeling of proteoliposomes containing MrpG subunits with an introduced cysteine in the C-terminal segment, the proteoliposomes (300 μl) were mixed with 0.1 mM MPB and dispensed as two aliquots (150 μl each).

    Article Title: Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding
    Article Snippet: Next 100 μl of an biotinylated anti-HSA antibody (Abcam) at 1.5 μg/ml in PBST was added to the plates followed incubation for 30 min. Plates were washed as above, and 100 μl of 1.25 μg/ml HRP-conjugated streptavidin (Sigma-Aldrich) in PBST was added to the plates and incubated for 30 min. .. Next 100 μl of an biotinylated anti-HSA antibody (Abcam) at 1.5 μg/ml in PBST was added to the plates followed incubation for 30 min. Plates were washed as above, and 100 μl of 1.25 μg/ml HRP-conjugated streptavidin (Sigma-Aldrich) in PBST was added to the plates and incubated for 30 min.

    Construct:

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: In addition, other polyclonal rabbit primary antibodies, such as E- cadherin (dilution = 1∶400, Cat.PA5-32178, Thermoscientific, IL), laminin 5 (dilution = 1∶400, Cat.ab14509, abcam, MA), β1 integrin (dilution = 1∶400, Cat.PA5–29606, Thermoscientific, IL) were incubated with 3D culture constructs overnight at 4°C and detected with FITC conjugated goat antibody (dilution = 1∶1000, Cat.65–111, Lifetech, CA). .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Enzyme-linked Immunospot:

    Article Title: Role of CD8+ Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
    Article Snippet: Paragraph title: Enzyme-linked immunospot (ELISPOT) assay. ... The cells were lysed, and the plates were developed with biotinylated rabbit, anti-rhesus gamma interferon antibody (BioSource International), streptavidin-alkaline phosphatase (Sigma, St. Louis, Mo), and BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md).

    Staining:

    Article Title: Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1? (MIP-1?)-dependent Pathways
    Article Snippet: All antibodies were diluted in PBS containing 5% normal serum and used at concentrations determined to give optimal staining intensities. .. Equivalent concentrations of rabbit, rat, goat or mouse IgG were used as control antibodies ( Sigma Chemical Co. ).

    Article Title: Tumor Bioengineering Using a Transglutaminase Crosslinked Hydrogel
    Article Snippet: After washing with PBS, cells were permeabilized with 0.5% Triton X-100/PBS, and blocked with 3% bovine serum albumin for 30 minutes at 37°C following by incubation with fluorescence protein conjugated primary antibody Rhodamine Phalloidin 33 nM (Cat.R415, Lifetech, CA) and DAPI dihydrochloride (dilution = 1∶100000, Cat.40011, Biotium Inc., CA) (nuclei staining) in the dark for 5 minutes. .. For immunocytochemistry, the samples were proceed with the same procedure as above and incubated with polyclonal rabbit primary antibody Ki67 (dilution = 1∶100, Cat.PA1–21520, Thermoscientific, IL), anti-rabbit IgG-biotin antibody from goat as secondary antibody (dilution = 1∶600, Cat.B8895, Sigma-Aldrich, MO), streptavidin-peroxisase polymer as signal amplifier (dilution = 1∶800, Cat.S2438, Sigma-Aldrich, MO) and detected by Pierce Peroxidase Detection Kit (Cat.36000, Thermo scientific, IL).

    Article Title: Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment
    Article Snippet: Anti-ubiquitin antibody (U5379), 6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS102, N179), (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK801, M107), 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466, G119), GSNO (N4148), dl -DTT (43817), Z-Leu-Leu-Leu-al (MG132, C2211), N -acetyl- l -cysteine (NAC, A7250), neocuproine (N1501), methyl methylthiomethyl sulfoxide (MMTS, 177954), (+)-sodium l -ascorbate (A7631), streptavidin-agarose (A1638), and Z-Leu-Leu-Glu β-naphthylamide (Z-Leu-Leu-Glu βNA, C0788) were purchased from Sigma-Aldrich (Poole, U.K.). .. Guava TUNEL kit (4500–0121) was purchased from Millipore Co. (Bedford, MA).

    Article Title: The Importance of LAT in the Activation, Homeostasis, and Regulatory Function of T Cells
    Article Snippet: Splenocytes were first loaded with Indo-1 acetoxymethyl ester (Molecular Probes) and then stained with phycoerythrin-conjugated anti-CD4 antibody. .. Calcium mobilization was initiated by the addition of biotin-anti-CD3ϵ (5 μg/ml) with biotin-anti-CD4 (1 μg/ml), followed by cross-linking with streptavidin (25 μg/ml, Sigma), and was monitored using FACStar (BD Biosciences).

    Article Title: c-Jun-dependent β3GnT8 promotes tumorigenesis and metastasis of hepatocellular carcinoma by inducing CD147 glycosylation and altering N-glycan patterns
    Article Snippet: Following 3 washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime, Haimen, China) at room temperature for 1 h. After additional 3 washes with TBST, the protein bands were visualized using an enhanced chemiluminescence (ECL) kit (GE Healthcare Life Sciences, Shanghai, China). .. Tissue microarray slides containing 75 pairs of liver cancer tissue and adjacent paracancer tissue arrays were purchased from Outdo Biotech (Shanghai, China) and stained with a rabbit anti-β3GnT8 antibody and HRP-conjugated anti-rabbit IgG secondary antibody (Beyotime, Haimen, China) or biotinylated-lycppersicon esculentum (Tomato) lectin (LEL) and HRP-conjugated streptavidin (Sigama, Sigma, St. Louis, MO, USA). .. A DAB peroxidase substrate kit (Beyotime, Haimen, China) was used for visualization of enzymatic reaction.

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Gels were stained with either Bio-Safe Coomassie blue (Bio-Rad), silver stain (Bio-Rad), or Schiff's reagent (Sigma) as recommended by the manufacturers. .. Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization.

    SDS Page:

    Article Title: p38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice
    Article Snippet: Protein extracts (10–25 μg/lane) were fractioned by 10% (w/v) SDS-PAGE and electroblotted onto an Immobilon-P membrane (Millipore, Billerica, MA). .. Membranes were incubated first with antibodies against phosphorylated TGF-β-activated kinase 1 (phospho-TAK1 (TGF-β-activated kinase 1)), phospho-Smad2, phospho-p38 MAPK, or phospho-ATF2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA) and subsequently with biotin-labeled anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) and horseradish peroxidase-conjugated Streptavidin (Millipore).

    Article Title: Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits Streptococcus mutans Glucosyltransferase
    Article Snippet: Paragraph title: SDS-PAGE and Western blot analysis. ... Biotinylated goat anti-rabbit IgG (Southern Biotechnology Associates) was used to develop Western blots probed with anti-α-amylase antibodies, whereas biotinylated rabbit anti-GLU was used to develop the Western blot probed with purified GLU; this was followed by incubation with alkaline phosphatase-conjugated streptavidin and by treatment with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium tablets (Sigma) for visualization.

    Article Title: Purification and Functional Reconstitution of a Seven-Subunit Mrp-Type Na+/H+ Antiporter
    Article Snippet: The Ni-NTA resin was washed with 10 column volumes of W2 buffer and eluted with 3 column volumes of E2 buffer. .. The eluted proteins were applied to gels for Tricine SDS-PAGE (10% acrylamide) ( ) and detected by Western blotting using either horseradish peroxidase (HRP)-conjugated anti-His-tag polyclonal antibodies (Abcam) or streptavidin-peroxidase polymer (Sigma-Aldrich). .. For MPB labeling of proteoliposomes containing MrpG subunits with an introduced cysteine in the C-terminal segment, the proteoliposomes (300 μl) were mixed with 0.1 mM MPB and dispensed as two aliquots (150 μl each).

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  • 99
    Millipore phosphatase conjugated streptavidin
    Characterization of wild-type and CD68 KO mice and their macrophages. (A) The genotype of the CD68 KO mice was confirmed using PCR with CD68-specific primers and mouse genomic DNA. +/+, wild-type mouse; +/−, heterozygous mouse; −/−, homozygous KO mouse. PCR with GAPDH primers served as a positive control. (B) Western blot of membrane proteins either stained (left) or incubated with an anti-CD68 antibody (right). Primary hepatocytes (Hep) served as a negative control. Arrow indicates CD68 signal in the +/+ lane. (C) Immunofluorescence assays of peritoneal macrophages from both wild-type and CD68 KO mice for anti-F4/80 expression. DIC, differential interference contrast. Bar, 20 µm. (D) Phagocytic activity of macrophages from CD68 KO mice. Flow cytometry results are shown for peritoneal macrophages incubated with two different fluorescent latex bead concentrations (Exp. 1 and Exp. 2 as indicated on the right of the figure). (E) KO macrophage recognition of the P39 peptide. Peritoneal macrophages from wild-type and CD68 KO mice were stained with the F4/80 antibody (recognizes macrophage-like cells) or an anti-CD68 antibody as indicated (top). The two cell types were also tested for uptake of the P39 peptide using the scrambled version, SC39, as a control (bottom). After incubation with the biotinylated peptides, cells were washed, fixed, and permeabilized for detection of intracellular peptide with Alexa Fluor 488–conjugated <t>streptavidin.</t> Data represent two independent experiments.
    Phosphatase Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphatase conjugated streptavidin/product/Millipore
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    94
    Millipore streptavidin alkaline phosphatase conjugate
    LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by <t>streptavidin-PE.</t>
    Streptavidin Alkaline Phosphatase Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin alkaline phosphatase conjugate/product/Millipore
    Average 94 stars, based on 15 article reviews
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    77
    Millipore streptavidin qds585 solution
    The fluorescence quenching efficiency of <t>QDs585-pep-BHQ-1</t> to <t>streptavidin-QDs585.</t> The graph shows the fluorescence quenching efficiency (%) of QDs585-pep-BHQ-1/streptavidin-QDs585 (3nM) at various additional concentrations (0–117 nM) of biotin-pep-BHQ-1.
    Streptavidin Qds585 Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of wild-type and CD68 KO mice and their macrophages. (A) The genotype of the CD68 KO mice was confirmed using PCR with CD68-specific primers and mouse genomic DNA. +/+, wild-type mouse; +/−, heterozygous mouse; −/−, homozygous KO mouse. PCR with GAPDH primers served as a positive control. (B) Western blot of membrane proteins either stained (left) or incubated with an anti-CD68 antibody (right). Primary hepatocytes (Hep) served as a negative control. Arrow indicates CD68 signal in the +/+ lane. (C) Immunofluorescence assays of peritoneal macrophages from both wild-type and CD68 KO mice for anti-F4/80 expression. DIC, differential interference contrast. Bar, 20 µm. (D) Phagocytic activity of macrophages from CD68 KO mice. Flow cytometry results are shown for peritoneal macrophages incubated with two different fluorescent latex bead concentrations (Exp. 1 and Exp. 2 as indicated on the right of the figure). (E) KO macrophage recognition of the P39 peptide. Peritoneal macrophages from wild-type and CD68 KO mice were stained with the F4/80 antibody (recognizes macrophage-like cells) or an anti-CD68 antibody as indicated (top). The two cell types were also tested for uptake of the P39 peptide using the scrambled version, SC39, as a control (bottom). After incubation with the biotinylated peptides, cells were washed, fixed, and permeabilized for detection of intracellular peptide with Alexa Fluor 488–conjugated streptavidin. Data represent two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: CD68 acts as a major gateway for malaria sporozoite liver infection

    doi: 10.1084/jem.20110575

    Figure Lengend Snippet: Characterization of wild-type and CD68 KO mice and their macrophages. (A) The genotype of the CD68 KO mice was confirmed using PCR with CD68-specific primers and mouse genomic DNA. +/+, wild-type mouse; +/−, heterozygous mouse; −/−, homozygous KO mouse. PCR with GAPDH primers served as a positive control. (B) Western blot of membrane proteins either stained (left) or incubated with an anti-CD68 antibody (right). Primary hepatocytes (Hep) served as a negative control. Arrow indicates CD68 signal in the +/+ lane. (C) Immunofluorescence assays of peritoneal macrophages from both wild-type and CD68 KO mice for anti-F4/80 expression. DIC, differential interference contrast. Bar, 20 µm. (D) Phagocytic activity of macrophages from CD68 KO mice. Flow cytometry results are shown for peritoneal macrophages incubated with two different fluorescent latex bead concentrations (Exp. 1 and Exp. 2 as indicated on the right of the figure). (E) KO macrophage recognition of the P39 peptide. Peritoneal macrophages from wild-type and CD68 KO mice were stained with the F4/80 antibody (recognizes macrophage-like cells) or an anti-CD68 antibody as indicated (top). The two cell types were also tested for uptake of the P39 peptide using the scrambled version, SC39, as a control (bottom). After incubation with the biotinylated peptides, cells were washed, fixed, and permeabilized for detection of intracellular peptide with Alexa Fluor 488–conjugated streptavidin. Data represent two independent experiments.

    Article Snippet: Specific antibody (anti–rat CD68 [AbD Serotec], anti–mouse and –human CD68 [eBioscience], and anti-gp96 [Sigma-Aldrich]) or peptide binding was visualized with alkaline phosphatase–conjugated secondary antibodies (Promega) or phosphatase-conjugated streptavidin (EMD Millipore), respectively.

    Techniques: Gene Knockout, Mouse Assay, Polymerase Chain Reaction, Positive Control, Western Blot, Staining, Incubation, Negative Control, Immunofluorescence, Expressing, Activity Assay, Flow Cytometry, Cytometry

    The P39 peptide binds to a macrophage-specific surface protein. (A) Primary Kupffer cells were incubated with heparinase I, chondroitinase ABC, or trypsin, as indicated. PBS buffer (no enzyme) served as a control. After enzyme treatment, 100 µg/ml biotinylated P39 peptide (green) and the anti-F4/80 antibody (red) were incubated for 1 h and visualized. The top row shows differential interference contrast (DIC) microscopy images merged with DAPI-stained nuclei (blue). P39 peptide binding was trypsin sensitive and unaffected by GAG-removing enzyme treatment. Bar, 20 µm. (B) Lysates from five different cell types were fractionated by gel electrophoresis, and binding of biotinylated P39 peptide to proteins on the blot was detected using alkaline phosphatase–conjugated streptavidin. KC, primary rat Kupffer cells; PtM, primary rat peritoneal macrophages; Raw, a mouse macrophage-like cell line; Hep, primary rat hepatocytes; ASM, primary rat airway smooth muscle cells. P39 peptide binding to an ∼110-kD protein band (arrows) was detected only for the macrophage-like cells (rat KC, rat PtM, and mouse Raw). (C) Kupffer cell fractions were tested for P39 peptide binding. M, membrane fraction; C, cytosolic fraction; I, insoluble fraction. P39 peptide bound only to an ∼110-kD membrane protein (arrow). (D) Activation of the human monocyte cell line THP-1 with PMA treatment for differentiation into a macrophage-like cell resulted in production of a protein recognized by the P39 peptide. No binding was detected for control THP-1 cells treated with DMSO carrier alone. An anti-actin antibody was used as a loading control. Immunofluorescence and far-Western blotting images are representative of two to three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: CD68 acts as a major gateway for malaria sporozoite liver infection

    doi: 10.1084/jem.20110575

    Figure Lengend Snippet: The P39 peptide binds to a macrophage-specific surface protein. (A) Primary Kupffer cells were incubated with heparinase I, chondroitinase ABC, or trypsin, as indicated. PBS buffer (no enzyme) served as a control. After enzyme treatment, 100 µg/ml biotinylated P39 peptide (green) and the anti-F4/80 antibody (red) were incubated for 1 h and visualized. The top row shows differential interference contrast (DIC) microscopy images merged with DAPI-stained nuclei (blue). P39 peptide binding was trypsin sensitive and unaffected by GAG-removing enzyme treatment. Bar, 20 µm. (B) Lysates from five different cell types were fractionated by gel electrophoresis, and binding of biotinylated P39 peptide to proteins on the blot was detected using alkaline phosphatase–conjugated streptavidin. KC, primary rat Kupffer cells; PtM, primary rat peritoneal macrophages; Raw, a mouse macrophage-like cell line; Hep, primary rat hepatocytes; ASM, primary rat airway smooth muscle cells. P39 peptide binding to an ∼110-kD protein band (arrows) was detected only for the macrophage-like cells (rat KC, rat PtM, and mouse Raw). (C) Kupffer cell fractions were tested for P39 peptide binding. M, membrane fraction; C, cytosolic fraction; I, insoluble fraction. P39 peptide bound only to an ∼110-kD membrane protein (arrow). (D) Activation of the human monocyte cell line THP-1 with PMA treatment for differentiation into a macrophage-like cell resulted in production of a protein recognized by the P39 peptide. No binding was detected for control THP-1 cells treated with DMSO carrier alone. An anti-actin antibody was used as a loading control. Immunofluorescence and far-Western blotting images are representative of two to three independent experiments.

    Article Snippet: Specific antibody (anti–rat CD68 [AbD Serotec], anti–mouse and –human CD68 [eBioscience], and anti-gp96 [Sigma-Aldrich]) or peptide binding was visualized with alkaline phosphatase–conjugated secondary antibodies (Promega) or phosphatase-conjugated streptavidin (EMD Millipore), respectively.

    Techniques: Incubation, Microscopy, Staining, Binding Assay, Nucleic Acid Electrophoresis, Activation Assay, Immunofluorescence, Far Western Blot

    Specificity of P39 peptide binding and inhibition of sporozoite entry. (A) A scrambled peptide with the same amino acid composition as P39 (SC39) was designed to serve as a negative control. All peptides have a cysteine residue (red) at positions 2 and 11. (B) The cell type indicated at the top of each panel was incubated with buffer alone (PBS), with a biotinylated P39 peptide (P39), or with a biotinylated scrambled peptide (SC39), followed by incubation with fluorescent streptavidin and analysis by flow cytometry. The P39 peptide bound to macrophage-like cells (Kupffer, PMA-activated THP-1) but not to hepatocytes (Hep). The scrambled peptide did not bind appreciably to any of the cell types. All images are representative of two to three independent experiments. (C and D) Inhibition of sporozoite entry in vitro. (C) Each cell type was incubated with PBS alone or with the P39 peptide, before exposure to 2 × 10 4 P. berghei sporozoites. The mean number of internalized sporozoites per 20 microscopic fields/well under 400-fold magnification was determined, and the percent inhibition relative to the PBS-treated group is shown at the bottom of the figure. (D) The SC39 peptide was tested for inhibition of P. berghei and P. falciparum sporozoite entry into each cell type. P39 peptide and PBS treatments were used as positive and negative controls, respectively. (C and D) Data from three independent biological experiments were combined. P-values (*, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD68 acts as a major gateway for malaria sporozoite liver infection

    doi: 10.1084/jem.20110575

    Figure Lengend Snippet: Specificity of P39 peptide binding and inhibition of sporozoite entry. (A) A scrambled peptide with the same amino acid composition as P39 (SC39) was designed to serve as a negative control. All peptides have a cysteine residue (red) at positions 2 and 11. (B) The cell type indicated at the top of each panel was incubated with buffer alone (PBS), with a biotinylated P39 peptide (P39), or with a biotinylated scrambled peptide (SC39), followed by incubation with fluorescent streptavidin and analysis by flow cytometry. The P39 peptide bound to macrophage-like cells (Kupffer, PMA-activated THP-1) but not to hepatocytes (Hep). The scrambled peptide did not bind appreciably to any of the cell types. All images are representative of two to three independent experiments. (C and D) Inhibition of sporozoite entry in vitro. (C) Each cell type was incubated with PBS alone or with the P39 peptide, before exposure to 2 × 10 4 P. berghei sporozoites. The mean number of internalized sporozoites per 20 microscopic fields/well under 400-fold magnification was determined, and the percent inhibition relative to the PBS-treated group is shown at the bottom of the figure. (D) The SC39 peptide was tested for inhibition of P. berghei and P. falciparum sporozoite entry into each cell type. P39 peptide and PBS treatments were used as positive and negative controls, respectively. (C and D) Data from three independent biological experiments were combined. P-values (*, P

    Article Snippet: Specific antibody (anti–rat CD68 [AbD Serotec], anti–mouse and –human CD68 [eBioscience], and anti-gp96 [Sigma-Aldrich]) or peptide binding was visualized with alkaline phosphatase–conjugated secondary antibodies (Promega) or phosphatase-conjugated streptavidin (EMD Millipore), respectively.

    Techniques: Binding Assay, Inhibition, Negative Control, Incubation, Flow Cytometry, Cytometry, In Vitro

    LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by streptavidin-PE.

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by streptavidin-PE.

    Article Snippet: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

    Techniques: Binding Assay, Expressing, Staining

    E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).

    Article Snippet: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

    Techniques: Concentration Assay, Binding Assay, Expressing, Staining, Lysis, Immunoprecipitation, SDS Page

    A novel mAb to TLR4-MD-2 reveals the LPS-triggered change of cell surface TLR4-MD-2. (a) Immunoprecipitation with anti-flag (top) or Sa15-21 (bottom) was conducted with Ba/F3 transfectants expressing the indicated molecules (Materials and Methods). The precipitates were probed with rabbit anti–mouse TLR4 sera followed by goat anti–rabbit alkaline phosphatase. Only immature, smaller TLR4 is detected in cells expressing TLR4 alone (top, TLR4f and CD14/TLR4f), because TLR4 without MD-2 cannot reach the cell surface. (b) Ba/F3 transfectants expressing CD14 and TLR4-MD-2 were stimulated with medium alone, 1 μg/ml lipid A, or 1 μg/ml LPS at 37°C for 30 min. Cells were stained with biotinylated MTS510 mAb or Sa15-21 as indicated, followed by streptavidin-PE. Open histograms depict staining with streptavidin-PE alone. (c) Ba/F3 cells expressing TLR4-MD-2 and CD14 (top) or RAW264 (bottom) were stimulated with medium, 2 μg/ml LPS, or 2 μg/ml lipid A antagonist E5531 as indicated at 37°C for 30 min. After washing, cells were subjected to cell surface biotinylation, detergent lysis, immunoprecipitation with MTS510 mAb (right three lanes) or Sa15-21 mAb (left three lanes), SDS-PAGE (7.5% polyacrylamide under nonreducing conditions), and electroblotting. Precipitated cell surface TLR4 was probed with streptavidin–alkaline phosphatase conjugate.

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: A novel mAb to TLR4-MD-2 reveals the LPS-triggered change of cell surface TLR4-MD-2. (a) Immunoprecipitation with anti-flag (top) or Sa15-21 (bottom) was conducted with Ba/F3 transfectants expressing the indicated molecules (Materials and Methods). The precipitates were probed with rabbit anti–mouse TLR4 sera followed by goat anti–rabbit alkaline phosphatase. Only immature, smaller TLR4 is detected in cells expressing TLR4 alone (top, TLR4f and CD14/TLR4f), because TLR4 without MD-2 cannot reach the cell surface. (b) Ba/F3 transfectants expressing CD14 and TLR4-MD-2 were stimulated with medium alone, 1 μg/ml lipid A, or 1 μg/ml LPS at 37°C for 30 min. Cells were stained with biotinylated MTS510 mAb or Sa15-21 as indicated, followed by streptavidin-PE. Open histograms depict staining with streptavidin-PE alone. (c) Ba/F3 cells expressing TLR4-MD-2 and CD14 (top) or RAW264 (bottom) were stimulated with medium, 2 μg/ml LPS, or 2 μg/ml lipid A antagonist E5531 as indicated at 37°C for 30 min. After washing, cells were subjected to cell surface biotinylation, detergent lysis, immunoprecipitation with MTS510 mAb (right three lanes) or Sa15-21 mAb (left three lanes), SDS-PAGE (7.5% polyacrylamide under nonreducing conditions), and electroblotting. Precipitated cell surface TLR4 was probed with streptavidin–alkaline phosphatase conjugate.

    Article Snippet: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

    Techniques: Immunoprecipitation, Expressing, Staining, Lysis, SDS Page

    The fluorescence quenching efficiency of QDs585-pep-BHQ-1 to streptavidin-QDs585. The graph shows the fluorescence quenching efficiency (%) of QDs585-pep-BHQ-1/streptavidin-QDs585 (3nM) at various additional concentrations (0–117 nM) of biotin-pep-BHQ-1.

    Journal:

    Article Title: Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application

    doi: 10.3727/215517915X689074

    Figure Lengend Snippet: The fluorescence quenching efficiency of QDs585-pep-BHQ-1 to streptavidin-QDs585. The graph shows the fluorescence quenching efficiency (%) of QDs585-pep-BHQ-1/streptavidin-QDs585 (3nM) at various additional concentrations (0–117 nM) of biotin-pep-BHQ-1.

    Article Snippet: The streptavidin-QDs585 solution was diluted to 3 nM with purified water (Millipore Milli-Q Lab Purification System; Tokyo; Japan).

    Techniques: Fluorescence

    The relative change in the fluorescence intensity rate and fluorescence lifetime decay of QDs585-pep-BHQ-1. (A) The relative changes in the fluorescence intensity rate ( I 0 / I ) of streptavidin-QDs585 ( I 0 ) (3 nM) and biotin-pep-BHQ-1 ( I ) (0–117

    Journal:

    Article Title: Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application

    doi: 10.3727/215517915X689074

    Figure Lengend Snippet: The relative change in the fluorescence intensity rate and fluorescence lifetime decay of QDs585-pep-BHQ-1. (A) The relative changes in the fluorescence intensity rate ( I 0 / I ) of streptavidin-QDs585 ( I 0 ) (3 nM) and biotin-pep-BHQ-1 ( I ) (0–117

    Article Snippet: The streptavidin-QDs585 solution was diluted to 3 nM with purified water (Millipore Milli-Q Lab Purification System; Tokyo; Japan).

    Techniques: Fluorescence

    The fluorescence images and fluorescence spectral changes of streptavdin-QDs585 and streptavidin-QDs585 conjugated with biotin-GPLGVRG[K(BHQ-1)]-CONH 2 (biotin-pep-BHQ-1). (A) The fluorescence images of streptavdin-QDs585 and QDs585-pep-BHQ-1. The fluorescence

    Journal:

    Article Title: Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application

    doi: 10.3727/215517915X689074

    Figure Lengend Snippet: The fluorescence images and fluorescence spectral changes of streptavdin-QDs585 and streptavidin-QDs585 conjugated with biotin-GPLGVRG[K(BHQ-1)]-CONH 2 (biotin-pep-BHQ-1). (A) The fluorescence images of streptavdin-QDs585 and QDs585-pep-BHQ-1. The fluorescence

    Article Snippet: The streptavidin-QDs585 solution was diluted to 3 nM with purified water (Millipore Milli-Q Lab Purification System; Tokyo; Japan).

    Techniques: Fluorescence