Structured Review

Agilent technologies biotinylated proteins
Evidence for a specific membrane antigen and its immunoprecipitation by MuSK-antibody positive IgG. a , three MuSK-MG plasmas displayed binding to TE671 cells (median fluorescence intensity) greater than the mean + 2 S.D. of the binding of control sera ( dotted line ). b , incubation of a <t>surface-biotinylated</t> TE671 cell extract with any one of three MuSK-MG plasmas resulted in the immunoprecipitation of a 90-kDa band of biotinylated protein. This band was not present when immunoprecipitation was performed using two healthy control subject sera or using a MuSK-seronegative MG patient plasma. c , the immunoprecipitation technique was refined to reduce nonspecific binding by incubation of intact TE671 cells with plasma followed by washing before cell extraction. The silver-stained gel contained a large number of protein bands matched between the patient, healthy control, and blank immunoprecipitates. On the blot, which was probed with HRP-conjugated streptavidin, there was a strong band at 90 kDa ( arrow ), which was exclusive to the MuSK-MG immunoprecipitate, and a band around 55 kDa present in all lanes ( arrowhead ) that must have been nonspecifically captured. d , a goat anti-MuSK antibody bound to a 90-kDa protein band derived from unbiotinylated TE671 cells, which had been immunoprecipitated using a MuSK-MG plasma. This band was of the same molecular mass as the band derived from surface-biotinylated TE671 cells, which had been immunoprecipitated using the same MuSK-MG plasma. HC , healthy control; + ve , positive.
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1) Product Images from "Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies *"

Article Title: Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M800563-MCP200

Evidence for a specific membrane antigen and its immunoprecipitation by MuSK-antibody positive IgG. a , three MuSK-MG plasmas displayed binding to TE671 cells (median fluorescence intensity) greater than the mean + 2 S.D. of the binding of control sera ( dotted line ). b , incubation of a surface-biotinylated TE671 cell extract with any one of three MuSK-MG plasmas resulted in the immunoprecipitation of a 90-kDa band of biotinylated protein. This band was not present when immunoprecipitation was performed using two healthy control subject sera or using a MuSK-seronegative MG patient plasma. c , the immunoprecipitation technique was refined to reduce nonspecific binding by incubation of intact TE671 cells with plasma followed by washing before cell extraction. The silver-stained gel contained a large number of protein bands matched between the patient, healthy control, and blank immunoprecipitates. On the blot, which was probed with HRP-conjugated streptavidin, there was a strong band at 90 kDa ( arrow ), which was exclusive to the MuSK-MG immunoprecipitate, and a band around 55 kDa present in all lanes ( arrowhead ) that must have been nonspecifically captured. d , a goat anti-MuSK antibody bound to a 90-kDa protein band derived from unbiotinylated TE671 cells, which had been immunoprecipitated using a MuSK-MG plasma. This band was of the same molecular mass as the band derived from surface-biotinylated TE671 cells, which had been immunoprecipitated using the same MuSK-MG plasma. HC , healthy control; + ve , positive.
Figure Legend Snippet: Evidence for a specific membrane antigen and its immunoprecipitation by MuSK-antibody positive IgG. a , three MuSK-MG plasmas displayed binding to TE671 cells (median fluorescence intensity) greater than the mean + 2 S.D. of the binding of control sera ( dotted line ). b , incubation of a surface-biotinylated TE671 cell extract with any one of three MuSK-MG plasmas resulted in the immunoprecipitation of a 90-kDa band of biotinylated protein. This band was not present when immunoprecipitation was performed using two healthy control subject sera or using a MuSK-seronegative MG patient plasma. c , the immunoprecipitation technique was refined to reduce nonspecific binding by incubation of intact TE671 cells with plasma followed by washing before cell extraction. The silver-stained gel contained a large number of protein bands matched between the patient, healthy control, and blank immunoprecipitates. On the blot, which was probed with HRP-conjugated streptavidin, there was a strong band at 90 kDa ( arrow ), which was exclusive to the MuSK-MG immunoprecipitate, and a band around 55 kDa present in all lanes ( arrowhead ) that must have been nonspecifically captured. d , a goat anti-MuSK antibody bound to a 90-kDa protein band derived from unbiotinylated TE671 cells, which had been immunoprecipitated using a MuSK-MG plasma. This band was of the same molecular mass as the band derived from surface-biotinylated TE671 cells, which had been immunoprecipitated using the same MuSK-MG plasma. HC , healthy control; + ve , positive.

Techniques Used: Immunoprecipitation, Binding Assay, Fluorescence, Incubation, Staining, Derivative Assay

Estimation of quantity of immunoprecipitated MuSK. a , the 90-kDa protein immunoprecipitated ( arrow ) after incubation of TE671 cells with a MuSK-MG plasma was stained with HRP-conjugated streptavidin along with known quantities of biotinylated BSA stained by HRP-conjugated streptavidin that run at 67 kDa ( arrowhead ). b , the quantity of immunoprecipitated MuSK was estimated by comparison of the integrated optical density of the 90-kDa band with that of the 67-kDa band. HC , healthy control; IOD , integrated optical density.
Figure Legend Snippet: Estimation of quantity of immunoprecipitated MuSK. a , the 90-kDa protein immunoprecipitated ( arrow ) after incubation of TE671 cells with a MuSK-MG plasma was stained with HRP-conjugated streptavidin along with known quantities of biotinylated BSA stained by HRP-conjugated streptavidin that run at 67 kDa ( arrowhead ). b , the quantity of immunoprecipitated MuSK was estimated by comparison of the integrated optical density of the 90-kDa band with that of the 67-kDa band. HC , healthy control; IOD , integrated optical density.

Techniques Used: Immunoprecipitation, Incubation, Staining

2) Product Images from "Functional Characterization of Human Cancer-Derived TRKB Mutations"

Article Title: Functional Characterization of Human Cancer-Derived TRKB Mutations

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016871

Identification of human cancer-derived TRKB point mutants and generation of cell systems. ( A ) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKB L138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKB P507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. ( B ) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. ( C ) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. ( D ) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).
Figure Legend Snippet: Identification of human cancer-derived TRKB point mutants and generation of cell systems. ( A ) Sequencing analysis of gDNA from NCI-H2009 cells harboring the TRKB L138F mutation (left panel) and from MDA-MB-435 cells harboring the TRKB P507L mutation (right panel). Asterisks (*) indicate the respective mutated bases. Black letters denote wild-type residues, red letters denote mutant residues. ( B ) Schematic overview over all cancer-derived TRKB point mutations analyzed in this study. LRM: Leucine-Rich Motif, Ig: Immunoglobulin-like domain, TM: transmembrane domain. Numbers indicate positions of amino acid residues. ( C ) Expression levels of mutant or wild-type TRKB in RIE-1 cells, analyzed on immunoblot (IB). Tubulin serves as loading control. Numbers represent quantification of TRKB signal, normalized to alpha-tubulin, and relative to Vector control. ( D ) Cell surface biotinylation assay showing that at least a significant fraction of all TRKB mutants localizes to the cell membrane. Total cell surface proteins were biotinylated with Sulfo-NHS-LC-Biotin, lysed and TRKB was immunoprecipitated (IP) with TRK antibody (C-14, C-13 for control). After gel electrophoresis, biotinylated TRKB was visualized with streptavidin-HRP and total TRKB with TRK antibody (C-14). All wild-type and mutant TRKB proteins became biotinylated (upper left panel). On the right hand side the specificity of the assay is demonstrated: biotin signal was only detected for full-length TRKB (upper right panel, second lane), but not for cytosolic, truncated TPR-TRKB [25] (third lane, expected at ∼50 kD), and not in the control IP (lane 4) or in the absence of Sulfo-NHS-LC-Biotin (lane 5). IP of total full-length and truncated TRKB is shown in bottom panels. Arrowheads indicate full-length TRKB, arrow indicates truncated TPR-TRKB (just below Ig heavy chains).

Techniques Used: Derivative Assay, Sequencing, Mutagenesis, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Cell Surface Biotinylation Assay, Immunoprecipitation, Nucleic Acid Electrophoresis

3) Product Images from "RGD-avidin-biotin pretargeting to ?v?3 integrin enhances the proapoptotic activity of TNF? related apoptosis inducing ligand (TRAIL)"

Article Title: RGD-avidin-biotin pretargeting to ?v?3 integrin enhances the proapoptotic activity of TNF? related apoptosis inducing ligand (TRAIL)

Journal: Apoptosis

doi: 10.1007/s10495-007-0166-5

Association of biotinylated TRAIL with RGDPEG-avidin. 96-well plates were coated overnight with 50 ng RGDPEG-avidin and incubated with indicated compounds for 2 h. TRAIL binding was assessed by anti-TRAIL immunodetection and expressed relative to the highest signal obtained with biotin-NHS-TRAIL. Experiments were performed in triplicate
Figure Legend Snippet: Association of biotinylated TRAIL with RGDPEG-avidin. 96-well plates were coated overnight with 50 ng RGDPEG-avidin and incubated with indicated compounds for 2 h. TRAIL binding was assessed by anti-TRAIL immunodetection and expressed relative to the highest signal obtained with biotin-NHS-TRAIL. Experiments were performed in triplicate

Techniques Used: Avidin-Biotin Assay, Incubation, Binding Assay, Immunodetection

Proapoptotic activity of RGDPEG-avidin:TRAIL complexes. Assays were performed in 96-well plates seeded with HUVEC that were subsequently incubated at room temperature with RGDPEG-avidin (10 μg/ml) and biotinylated TRAIL (100 ng/ml), to allow assembly of the complexes. Next, Jurkat T cells were added followed by 4 h of incubation at 37°C, after which the activation of caspase 3/7 was determined by a luminescence based assay. Experiments were carried out in the absence of RGDPEG-avidin (control experiment, white bars), in the presence of excess of RGDPEG-avidin (striped bars, 100-fold molar excess over biotinylated TRAIL) or in wells incubated with RGDPEG-avidin that had been washed before addition of biotin-TRAIL and Jurkat cells (black bars). * P
Figure Legend Snippet: Proapoptotic activity of RGDPEG-avidin:TRAIL complexes. Assays were performed in 96-well plates seeded with HUVEC that were subsequently incubated at room temperature with RGDPEG-avidin (10 μg/ml) and biotinylated TRAIL (100 ng/ml), to allow assembly of the complexes. Next, Jurkat T cells were added followed by 4 h of incubation at 37°C, after which the activation of caspase 3/7 was determined by a luminescence based assay. Experiments were carried out in the absence of RGDPEG-avidin (control experiment, white bars), in the presence of excess of RGDPEG-avidin (striped bars, 100-fold molar excess over biotinylated TRAIL) or in wells incubated with RGDPEG-avidin that had been washed before addition of biotin-TRAIL and Jurkat cells (black bars). * P

Techniques Used: Activity Assay, Avidin-Biotin Assay, Incubation, Activation Assay, Luminescence Assay

Schematic representation of RGD-avidin:TRAIL complexes. Soluble recombinant human TRAIL was modified with biotin groups via two different reagents that either react at lysine or methionine residues. Biotinylated TRAIL was complexed to an avidin-based targeting device equipped with approximately 5 RGD-peptide ligands per avidin molecule. A hydrophilic PEG-linker was used to append the RGD-peptides to avidin. The assembled complexes can bind to tumor blood vessels via RGD//α v β 3 -integrin interactions, while apoptosis can be induced via TRAIL/DR ligation
Figure Legend Snippet: Schematic representation of RGD-avidin:TRAIL complexes. Soluble recombinant human TRAIL was modified with biotin groups via two different reagents that either react at lysine or methionine residues. Biotinylated TRAIL was complexed to an avidin-based targeting device equipped with approximately 5 RGD-peptide ligands per avidin molecule. A hydrophilic PEG-linker was used to append the RGD-peptides to avidin. The assembled complexes can bind to tumor blood vessels via RGD//α v β 3 -integrin interactions, while apoptosis can be induced via TRAIL/DR ligation

Techniques Used: Avidin-Biotin Assay, Recombinant, Modification, Ligation

Characterization of biotinylated TRAILs. Panel A: Anti-biotin ELISA with streptavidin-HRP. Signals were corrected for background and expressed relative to the maximum detected intensity. Panel B: MALDI-TOF analysis of TRAIL and biotin-NHS-TRAIL
Figure Legend Snippet: Characterization of biotinylated TRAILs. Panel A: Anti-biotin ELISA with streptavidin-HRP. Signals were corrected for background and expressed relative to the maximum detected intensity. Panel B: MALDI-TOF analysis of TRAIL and biotin-NHS-TRAIL

Techniques Used: Enzyme-linked Immunosorbent Assay

4) Product Images from "Were ancestral proteins less specific?"

Article Title: Were ancestral proteins less specific?

Journal: bioRxiv

doi: 10.1101/2020.05.27.120261

Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. Biotinylated, Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is
Figure Legend Snippet: Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. Biotinylated, Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is

Techniques Used: Binding Assay

5) Product Images from "In situ detection of protein interactions for recombinant therapeutic enzymes"

Article Title: In situ detection of protein interactions for recombinant therapeutic enzymes

Journal: bioRxiv

doi: 10.1101/2020.05.06.081885

Expression of bait-BirA proteins results in a substantial increase in biotinylated proteins. a) Successful secretion of the bait-BirA proteins into the culture supernatant was evaluated by Western blot using HRP-anti-flag antibody. b) The immunoblotting biotinylation profiling of the model proteins and WT control in HEK293 cells with HRP-streptavidin. When the BirA domain was fused to the model proteins, biotin addition led to the biotinylation of a subset of proteins (B+) which are not seen in WT or absence of biotin. This demonstrates that the BioID labeling system tags interactions as secreted proteins are synthesized and trafficked through the secretory pathway. A few endogenously biotinylated proteins appear in the absence of biotin and in the WT.
Figure Legend Snippet: Expression of bait-BirA proteins results in a substantial increase in biotinylated proteins. a) Successful secretion of the bait-BirA proteins into the culture supernatant was evaluated by Western blot using HRP-anti-flag antibody. b) The immunoblotting biotinylation profiling of the model proteins and WT control in HEK293 cells with HRP-streptavidin. When the BirA domain was fused to the model proteins, biotin addition led to the biotinylation of a subset of proteins (B+) which are not seen in WT or absence of biotin. This demonstrates that the BioID labeling system tags interactions as secreted proteins are synthesized and trafficked through the secretory pathway. A few endogenously biotinylated proteins appear in the absence of biotin and in the WT.

Techniques Used: Expressing, Western Blot, Labeling, Synthesized

Dozens of proteins show significantly increased biotinylation after expression of bait-BirA proteins. Volcano plot showing the distribution of the quantified biotinylated proteins by MS according to p-value and fold change. As depicted the bait-protein significantly showed the highest fold change compared to WT almost in all cases, indicating the capability of the BioID labeling system to tag the in vivo interactors within the live cells. SeAP is a truncated form of Alkaline phosphatase, placental type (ALPP).
Figure Legend Snippet: Dozens of proteins show significantly increased biotinylation after expression of bait-BirA proteins. Volcano plot showing the distribution of the quantified biotinylated proteins by MS according to p-value and fold change. As depicted the bait-protein significantly showed the highest fold change compared to WT almost in all cases, indicating the capability of the BioID labeling system to tag the in vivo interactors within the live cells. SeAP is a truncated form of Alkaline phosphatase, placental type (ALPP).

Techniques Used: Expressing, Labeling, In Vivo

6) Product Images from "Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3"

Article Title: Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001925

Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .
Figure Legend Snippet: Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .

Techniques Used: Western Blot, Staining, Flow Cytometry

7) Product Images from "Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling"

Article Title: Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

Journal: eLife

doi: 10.7554/eLife.42037

Determination of the coronavirus RTC-proximal proteome. ( a ) Schematic overview of the BirA R118G -mediated proximity biotinylation assay using MHV-BirA R118G -nsp2. ( b ) Western blot analysis of MHV-BirA R118G -nsp2-infected L929 cells. L929 cells were infected with MHV-BirA R118G -nsp2, MHV-A59 or non-infected in medium with and without supplementation of 67 µM biotin. Cells were lysed 15 h.p.i. and biotinylated factors were subjected to affinity purification using streptavidin-coupled magnetic beads. Total cell lysates and affinity-purified fractions were separated by SDS-PAGE and analysed by western blot probed with horse radish peroxidase (HRP)-coupled Streptavidin. ( c ) Host and viral factors identified by LC-MS/MS. 4*10 7 L929 cells were infected with MHV-BirA R118G -nsp2 or MHV-A59 in medium supplemented with 67 µM biotin. 15 h.p.i., lysates were affinity purified and LC-MS/MS was performed from in-gel digested samples. MS identification of biotinylated proteins was performed in three independent biological replicates. Spectral interpretation was performed against a Mus musculus and MHV database and log 2 -transformed LFQ levels (x-axis) were used to determine significant differences in protein enrichment between sample groups (Student's T-test, y-axis). Identified cellular proteins are displayed as black dots, MHV proteins are highlighted in red (nsp: non-structural protein, N: nucleocapsid, S: spike, M: membrane, 2a: accessory protein 2a). ( d ) Summary of viral proteins identified by LC-MS/MS. nsp2-10, nsp12-16, and nucleocapsid were significantly enriched in fractions derived from MHV-BirA R118G -nsp2-infected cells whereas nsp1, nsp11, structural proteins spike ( S ), envelope ( E ) and membrane proteins ( M ) as well as all accessory proteins (NS2a, HE, ORF4, ORF5a) were either not significantly enriched or not detected. ( e,f ) Immunofluorescence analysis of RTC-proximal cellular factors. L929 cells were seeded on coverslips, infected with MHV-BirA R118G -nsp2 ( e ) or MHV-A59 ( f ), fixed at 9 h.p.i. and processed for immunofluorescence using anti-myc, anti-RTN4 and anti-eIF3E antibodies ( e ) or anti-dsRNA, anti-RTN4 and anti-eIF3E antibodies ( f ). Secondary fluorophore-coupled antibodies were used to detect the viral replicase and endogenous levels of RTN4 and eIF3E ( e ). Scale bars: 10 µm; insets 5 µm. Proximity ligations were performed using Duolink In Situ detection reagents ( f ). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Intensity profiles highlighted in the magnified regions are shown. Scale bars: 20 µm (insets 5 µm).
Figure Legend Snippet: Determination of the coronavirus RTC-proximal proteome. ( a ) Schematic overview of the BirA R118G -mediated proximity biotinylation assay using MHV-BirA R118G -nsp2. ( b ) Western blot analysis of MHV-BirA R118G -nsp2-infected L929 cells. L929 cells were infected with MHV-BirA R118G -nsp2, MHV-A59 or non-infected in medium with and without supplementation of 67 µM biotin. Cells were lysed 15 h.p.i. and biotinylated factors were subjected to affinity purification using streptavidin-coupled magnetic beads. Total cell lysates and affinity-purified fractions were separated by SDS-PAGE and analysed by western blot probed with horse radish peroxidase (HRP)-coupled Streptavidin. ( c ) Host and viral factors identified by LC-MS/MS. 4*10 7 L929 cells were infected with MHV-BirA R118G -nsp2 or MHV-A59 in medium supplemented with 67 µM biotin. 15 h.p.i., lysates were affinity purified and LC-MS/MS was performed from in-gel digested samples. MS identification of biotinylated proteins was performed in three independent biological replicates. Spectral interpretation was performed against a Mus musculus and MHV database and log 2 -transformed LFQ levels (x-axis) were used to determine significant differences in protein enrichment between sample groups (Student's T-test, y-axis). Identified cellular proteins are displayed as black dots, MHV proteins are highlighted in red (nsp: non-structural protein, N: nucleocapsid, S: spike, M: membrane, 2a: accessory protein 2a). ( d ) Summary of viral proteins identified by LC-MS/MS. nsp2-10, nsp12-16, and nucleocapsid were significantly enriched in fractions derived from MHV-BirA R118G -nsp2-infected cells whereas nsp1, nsp11, structural proteins spike ( S ), envelope ( E ) and membrane proteins ( M ) as well as all accessory proteins (NS2a, HE, ORF4, ORF5a) were either not significantly enriched or not detected. ( e,f ) Immunofluorescence analysis of RTC-proximal cellular factors. L929 cells were seeded on coverslips, infected with MHV-BirA R118G -nsp2 ( e ) or MHV-A59 ( f ), fixed at 9 h.p.i. and processed for immunofluorescence using anti-myc, anti-RTN4 and anti-eIF3E antibodies ( e ) or anti-dsRNA, anti-RTN4 and anti-eIF3E antibodies ( f ). Secondary fluorophore-coupled antibodies were used to detect the viral replicase and endogenous levels of RTN4 and eIF3E ( e ). Scale bars: 10 µm; insets 5 µm. Proximity ligations were performed using Duolink In Situ detection reagents ( f ). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Intensity profiles highlighted in the magnified regions are shown. Scale bars: 20 µm (insets 5 µm).

Techniques Used: Cell Surface Biotinylation Assay, Western Blot, Infection, Affinity Purification, Magnetic Beads, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transformation Assay, Protein Enrichment, Derivative Assay, Immunofluorescence, In Situ, Imaging

Immunofluorescence analysis of the MHV-BirA R118G -nsp2 RTC and BirA R118G -nsp2 –mediated biotinylataion. MHV-BirA R118G -nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 12 hr post-infection (h.p.i.) and processed for immunofluorescence analysis with antibodies directed against the BirA R118G (anti-myc), the viral replicase (anti-nsp2/3, nsp8) and biotinylated factors (streptavidin). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Scale bars: 10 µm; insets 5 µm.
Figure Legend Snippet: Immunofluorescence analysis of the MHV-BirA R118G -nsp2 RTC and BirA R118G -nsp2 –mediated biotinylataion. MHV-BirA R118G -nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 12 hr post-infection (h.p.i.) and processed for immunofluorescence analysis with antibodies directed against the BirA R118G (anti-myc), the viral replicase (anti-nsp2/3, nsp8) and biotinylated factors (streptavidin). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Scale bars: 10 µm; insets 5 µm.

Techniques Used: Immunofluorescence, Infection, Cell Culture, Imaging

Immunofluorescence analysis of MHV-BirA R118G -nsp2-mediated biotinylation. MHV-BirA R118G -nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 9 and 15 hr post infection (h.p.i.) and processed for immunofluorescence analysis with antibodies directed against the BirA R118G (anti-myc), the viral replicase (anti-nsp2/3) and biotinylated factors (streptavidin). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Scale bars: 10 µm; insets 5 µm.
Figure Legend Snippet: Immunofluorescence analysis of MHV-BirA R118G -nsp2-mediated biotinylation. MHV-BirA R118G -nsp2, MHV-A59- or non-infected L929 fibroblasts were cultured in medium supplemented with 67 µM biotin. Cells were fixed 9 and 15 hr post infection (h.p.i.) and processed for immunofluorescence analysis with antibodies directed against the BirA R118G (anti-myc), the viral replicase (anti-nsp2/3) and biotinylated factors (streptavidin). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Scale bars: 10 µm; insets 5 µm.

Techniques Used: Immunofluorescence, Infection, Cell Culture, Imaging

Related Articles

Mutagenesis:

Article Title: Were ancestral proteins less specific?
Article Snippet: .. Preparation of biotinylated proteins for phage display A mutant version of hA5 with a single N-terminal Cys residues were generated via sitedirected mutagenesis using the QuikChange lightning system (Agilent). ..

Incubation:

Article Title: Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies *
Article Snippet: .. To immunostain bands of biotinylated proteins, filters were incubated in HRP-conjugated streptavidin (Dako catalog number P0397) diluted 1:10,000 in PBS Tween for 45 min at room temperature, washed in PBS Tween, and stained with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) color reagent (0.5 mg/ml DAB in PBS with 0.03% hydrogen peroxide) for between 5 and 15 min. To immunostain specifically for MuSK, the filter was incubated in goat polyclonal anti-MuSK extracellular domain antibody (R & D Systems catalog number AF562) diluted 1:200 in PBS Tween for 45 min at room temperature, washed in PBS Tween, and incubated in HRP-conjugated anti-goat immunoglobulins secondary antibody (Dako catalog number P0449) diluted 1:1000 in PBS Tween for 30 min at room temperature before DAB color development. .. Digestion and Preparation of Immunoprecipitation Products Prior to Liquid Chromatography and Mass Spectrometry Immunoprecipitation products, eluted in glycine-HCl and neutralized as described above, were lyophilized in a vacuum centrifuge.

Article Title: Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes
Article Snippet: .. Biotinylated proteins captured by the membrane-bound antibodies were detected by incubation with HRP-streptavidin and analysis was performed by an Agilent laser scanner (Agilent Technologies, Palo Alto, CA, USA). .. Spot intensities were quantified with ScanAlyze software (Michael Eisen, http://rana.lbl.gov/EisenSoftware.htm ) and mean signal and median background values were applied in subsequent calculations.

Article Title: RGD-avidin-biotin pretargeting to ?v?3 integrin enhances the proapoptotic activity of TNF? related apoptosis inducing ligand (TRAIL)
Article Snippet: .. Wells were coated with serial dilutions of the biotinylated proteins for 1 h at room temperature, washed with PBS containing 0.05% Tween20 and incubated for 1 h at room temperature with streptavidin-peroxidase complex (Dako, 1:2500 in PBS), followed by a standard incubation with OPD. .. The protein concentration at which 50% of the maximum absorbance was measured (EC50) was calculated by nonlinear regression (Graphpad Prism), and used to calculate relative biotin:protein ratios in each conjugate.

Article Title: Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling
Article Snippet: .. Membranes were incubated in a protein-free blocking buffer (Advansta) and biotinylated proteins were probed by incubation with horseradish peroxidase-conjugated Streptavidin (Dako). .. Proteins were visualized using WesternBright enhanced chemiluminescence horseradish peroxidase substrate (Advansta) according to the manufacturer's protocol.

Blocking Assay:

Article Title: Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling
Article Snippet: .. Membranes were incubated in a protein-free blocking buffer (Advansta) and biotinylated proteins were probed by incubation with horseradish peroxidase-conjugated Streptavidin (Dako). .. Proteins were visualized using WesternBright enhanced chemiluminescence horseradish peroxidase substrate (Advansta) according to the manufacturer's protocol.

Generated:

Article Title: Were ancestral proteins less specific?
Article Snippet: .. Preparation of biotinylated proteins for phage display A mutant version of hA5 with a single N-terminal Cys residues were generated via sitedirected mutagenesis using the QuikChange lightning system (Agilent). ..

Staining:

Article Title: Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies *
Article Snippet: .. To immunostain bands of biotinylated proteins, filters were incubated in HRP-conjugated streptavidin (Dako catalog number P0397) diluted 1:10,000 in PBS Tween for 45 min at room temperature, washed in PBS Tween, and stained with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) color reagent (0.5 mg/ml DAB in PBS with 0.03% hydrogen peroxide) for between 5 and 15 min. To immunostain specifically for MuSK, the filter was incubated in goat polyclonal anti-MuSK extracellular domain antibody (R & D Systems catalog number AF562) diluted 1:200 in PBS Tween for 45 min at room temperature, washed in PBS Tween, and incubated in HRP-conjugated anti-goat immunoglobulins secondary antibody (Dako catalog number P0449) diluted 1:1000 in PBS Tween for 30 min at room temperature before DAB color development. .. Digestion and Preparation of Immunoprecipitation Products Prior to Liquid Chromatography and Mass Spectrometry Immunoprecipitation products, eluted in glycine-HCl and neutralized as described above, were lyophilized in a vacuum centrifuge.

Affinity Purification:

Article Title: In situ detection of protein interactions for recombinant therapeutic enzymes
Article Snippet: .. Affinity purification of biotinylated proteins was carried out in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent). .. Briefly, cartridges were first primed with lysis buffer.

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  • 92
    Agilent technologies biotinylated proteins
    Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. <t>Biotinylated,</t> Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is
    Biotinylated Proteins, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated proteins/product/Agilent technologies
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    biotinylated proteins - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    93
    Agilent technologies serum free solution
    Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. <t>Biotinylated,</t> Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is
    Serum Free Solution, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/serum free solution/product/Agilent technologies
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    serum free solution - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. Biotinylated, Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is

    Journal: bioRxiv

    Article Title: Were ancestral proteins less specific?

    doi: 10.1101/2020.05.27.120261

    Figure Lengend Snippet: Set of binding peptides can be estimated using phage display. Rows show two different experiments, done in parallel, for each protein. Biotinylated, Ca 2+ -loaded, S100 is added to a population of phage either alone (row A) or with saturating competitor peptide added in trans (row B). Phage that bind to the protein (blue or purple) are pulled down using a streptavidin plate. Bound phage are then eluted using EDTA, which disrupts the peptide binding interface. In the absence of competitor (row A), phage bind adventitiously (purple) as well as at the interface of interest (blue). In the presence of competitor (row B), only adventitious binders are present. C) Distribution of enrichment values for peptides taken from pooled biological replicates of hA5. The measured distribution (gray) can be fit by the sum of two Gaussian distributions: responsive (blue) and unresponsive (purple), which sum to the total (yellow). The dashed line indicates cutoff for E values above which the probability the value arose from the unresponsive distribution is

    Article Snippet: Preparation of biotinylated proteins for phage display A mutant version of hA5 with a single N-terminal Cys residues were generated via sitedirected mutagenesis using the QuikChange lightning system (Agilent).

    Techniques: Binding Assay

    Expression of bait-BirA proteins results in a substantial increase in biotinylated proteins. a) Successful secretion of the bait-BirA proteins into the culture supernatant was evaluated by Western blot using HRP-anti-flag antibody. b) The immunoblotting biotinylation profiling of the model proteins and WT control in HEK293 cells with HRP-streptavidin. When the BirA domain was fused to the model proteins, biotin addition led to the biotinylation of a subset of proteins (B+) which are not seen in WT or absence of biotin. This demonstrates that the BioID labeling system tags interactions as secreted proteins are synthesized and trafficked through the secretory pathway. A few endogenously biotinylated proteins appear in the absence of biotin and in the WT.

    Journal: bioRxiv

    Article Title: In situ detection of protein interactions for recombinant therapeutic enzymes

    doi: 10.1101/2020.05.06.081885

    Figure Lengend Snippet: Expression of bait-BirA proteins results in a substantial increase in biotinylated proteins. a) Successful secretion of the bait-BirA proteins into the culture supernatant was evaluated by Western blot using HRP-anti-flag antibody. b) The immunoblotting biotinylation profiling of the model proteins and WT control in HEK293 cells with HRP-streptavidin. When the BirA domain was fused to the model proteins, biotin addition led to the biotinylation of a subset of proteins (B+) which are not seen in WT or absence of biotin. This demonstrates that the BioID labeling system tags interactions as secreted proteins are synthesized and trafficked through the secretory pathway. A few endogenously biotinylated proteins appear in the absence of biotin and in the WT.

    Article Snippet: Affinity purification of biotinylated proteins was carried out in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent).

    Techniques: Expressing, Western Blot, Labeling, Synthesized

    Dozens of proteins show significantly increased biotinylation after expression of bait-BirA proteins. Volcano plot showing the distribution of the quantified biotinylated proteins by MS according to p-value and fold change. As depicted the bait-protein significantly showed the highest fold change compared to WT almost in all cases, indicating the capability of the BioID labeling system to tag the in vivo interactors within the live cells. SeAP is a truncated form of Alkaline phosphatase, placental type (ALPP).

    Journal: bioRxiv

    Article Title: In situ detection of protein interactions for recombinant therapeutic enzymes

    doi: 10.1101/2020.05.06.081885

    Figure Lengend Snippet: Dozens of proteins show significantly increased biotinylation after expression of bait-BirA proteins. Volcano plot showing the distribution of the quantified biotinylated proteins by MS according to p-value and fold change. As depicted the bait-protein significantly showed the highest fold change compared to WT almost in all cases, indicating the capability of the BioID labeling system to tag the in vivo interactors within the live cells. SeAP is a truncated form of Alkaline phosphatase, placental type (ALPP).

    Article Snippet: Affinity purification of biotinylated proteins was carried out in a Bravo AssayMap platform (Agilent) using AssayMap streptavidin cartridges (Agilent).

    Techniques: Expressing, Labeling, In Vivo