streptavidin hrp reagent  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin hrp reagent
    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific <t>streptavidin</t> binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase <t>(HRP),</t> streptavidin (SA), primary antibody (1° Ab)
    Streptavidin Hrp Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp reagent/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp reagent - by Bioz Stars, 2020-03
    90/100 stars

    Images

    1) Product Images from "EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES"

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES

    Journal: Journal of immunological methods

    doi: 10.1016/j.jim.2018.10.006

    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)
    Figure Legend Snippet: Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)

    Techniques Used: Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry

    2) Product Images from "EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES"

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES

    Journal: Journal of immunological methods

    doi: 10.1016/j.jim.2018.10.006

    Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)
    Figure Legend Snippet: Peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (A) western blot and (B) ELISA using wells coated with streptavidin. (C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 μm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b-SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)

    Techniques Used: Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry

    Related Articles

    Blocking Assay:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: Blots were then washed extensively with PBST and incubated in block with secondary HRP-conjugated goat antibodies specific to rat IgG (H+L) or mouse IgG (H+L) (Thermo Scientific Fisher) for 1 hr at RT. .. In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

    Incubation:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: Blots were then washed extensively with PBST and incubated in block with secondary HRP-conjugated goat antibodies specific to rat IgG (H+L) or mouse IgG (H+L) (Thermo Scientific Fisher) for 1 hr at RT. .. In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

    Imaging:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: Washed blots were finally developed with a SignalFire ECL reagent (Cell Signaling, Danvers, MA) and exposed/imaged on a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA). .. In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

    Western Blot:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: Paragraph title: SDS-PAGE and western blot ... In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

    Binding Assay:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: .. In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential. .. A Pierce Classic IP kit (Thermo Scientific Fisher) was used to investigate ligand binding of soluble peptide/MHC protein.

    SDS Page:

    Article Title: EXPRESSION AND CHARACTERIZATION OF SOLUBLE EPITOPE-DEFINED MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) FROM STABLE EUKARYOTIC CELL LINES
    Article Snippet: Paragraph title: SDS-PAGE and western blot ... In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.

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    Thermo Fisher conjugated streptavidin
    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by <t>PE-streptavidin</t> and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P
    Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    conjugated streptavidin - by Bioz Stars, 2020-03
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    99
    Thermo Fisher streptavidin hrp
    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with <t>streptavidin</t> beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using <t>streptavidin-HRP</t> (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 162 article reviews
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    99
    Thermo Fisher streptavidin hrp conjugate
    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. <t>SAv-HRP,</t> <t>streptavidin–horseradish</t> peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp conjugate/product/Thermo Fisher
    Average 99 stars, based on 222 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


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

    Journal: Journal of Leukocyte Biology

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

    doi: 10.1189/jlb.2A0414-193RR

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

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

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

    Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Journal: Genes & Development

    Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

    doi: 10.1101/gad.305201.117

    Figure Lengend Snippet: Characterization of the BRWD2/PHIP CryptoTudor domain. ( A ) Diagram depicting the recombinant BRWD2/PHIP CryptoTudor–bromodomain (BRWD2/PHIP–Crypt–bromo) construct used for binding studies. N-terminal 10X-Histidine (red box) and C-terminal 1XFlag (green box) tags were included for protein purification. ( B ) Coomassie stained SDS-PAGE gel of in vitro chromatin capture experiment using the BRWD2/PHIP–Crypt–bromo module. (Lane 1 ) Input MNase-digested chromatin. (Lane 2 ) Chromatin subjected to anti-Flag immunoprecipitation without BRWD2/PHIP–Crypt–bromo. (Lane 3 ) Chromatin incubated with recombinant BRWD2/PHIP–Crypt–bromo and subjected to anti-Flag immunoprecipitation. ( C ) Quantitative MS analysis of histones captured in B . Bars indicate the fraction of total histone represented by each modification state in the input (gray) or Flag immunoprecipitation material (red). (Un) Unmodified; (me1) monomethylated; (me2) dimethylated; (me3) trimethylated; (Ac) acetylated. ( D ) Coomassie-stained SDS-PAGE gel of histone peptide pull-downs performed with human ( top ) or Drosophila ( bottom ) Crypt–bromo constructs. Recombinant protein was incubated with streptavidin beads alone (SA-beads) or the biotinylated histone peptides indicated. Ten percent of input and 20% of each pull-down sample were loaded. ( Bottom ) Eluted proteins were subjected to dot blotting using streptavidin-HRP (SA-HRP). ( E ) ITC experiments with human BRWD2–Crypt–bromo titrated with H3 unmodified ( left ), H3K4me1 ( middle ), and H3K4me3 ( right ) peptides. ( F ) Coomassie-stained SDS-PAGE gel of pull-downs performed with human BRWD2/PHIP–Crypt–bromo and a panel of histone peptides. Ten percent of input and 20% of each pull-down sample were loaded. Streptavidin-HRP dot blot loading control is shown below the gel image. ( G ) Coomassie-stained SDS-PAGE gel of a panel of histone peptide pull-downs performed with human BRWD2–Crypt–bromo ( top ), the isolated CryptoTudor domain ( middle ), or the isolated tandem bromodomains ( bottom ). Ten percent of input and 20% of each pull-down sample were loaded. Dot blot loading control is shown below each gel image. ( H ) Coomassie-stained gels of histone peptide pull-downs performed with point mutations of the BRWD2 CryptoTudor domain. Dot blot loading controls are shown below the gels. ( I ). Positions of residues predicted to be involved in methyl-lysine binding are highlighted in red.

    Article Snippet: As a loading control, 0.25 µL of the eluted proteins was spotted onto a nitrocellulose membrane and detected using streptavidin-HRP (Thermo Scientific, no. 21130).

    Techniques: Recombinant, Construct, Binding Assay, Protein Purification, Staining, SDS Page, In Vitro, Immunoprecipitation, Incubation, Mass Spectrometry, Modification, Dot Blot, Isolation

    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Journal: Nature Communications

    Article Title: Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

    doi: 10.1038/s41467-018-04343-0

    Figure Lengend Snippet: Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Article Snippet: The samples were analysed by western blotting using Streptavidin-HRP conjugate (SAv-HRP, Thermo, S911, 1:5000) and Coomassie Brilliant Blue (CBB) stain.

    Techniques: Western Blot, Recombinant, Incubation, Staining, Concentration Assay, Fluorescence

    Restoring protein synthesis delays retinal degeneration. (A) Protein synthesis levels depicted by the detection of azidohomoalanine incorporation into proteins with HRP-conjugated streptavidin and (B) coomassie staining for normalization of the membrane. (C) Graph showing relative levels of protein synthesis in the retinas of C57BL/6J (n = 3), rd16 (n = 3), and rd16 4ebp1/2 −/− (n = 3) mice at P15. (D) ERG waveforms of three groups and (E) graph showing mean ERG a- and b- wave amplitudes of C57BL/6J (n = 4), rd16 (n = 6), and rd16 4ebp1/2 −/− (n = 6) mice at P17. (F) Representative images of H E-stained retinal sections taken at P18 in the regions denoted as * in (G). (G) Spider plots depicting number of photoreceptor nuclei in the ONL as counted by a masked investigator. The spidergram was generated by plotting the number of nuclei using 200 μm step in the distance from the ONH for both hemispheres. (H) TUNEL staining of retinal sections from C57BL/6J (n = 3), rd16 (n = 4), and rd16 4ebp1/2 −/− (n = 3) eyes at P15. (I) Graph showing TUNEL-positive nuclei per square millimeter in the three groups. Green, TUNEL; blue, DAPI; a.u, arbitrary units; kD, kilodaltons. Error bars: SEM. Statistical significance denoted by *P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Role of Translational Attenuation in Inherited Retinal Degeneration

    doi: 10.1167/iovs.19-27512

    Figure Lengend Snippet: Restoring protein synthesis delays retinal degeneration. (A) Protein synthesis levels depicted by the detection of azidohomoalanine incorporation into proteins with HRP-conjugated streptavidin and (B) coomassie staining for normalization of the membrane. (C) Graph showing relative levels of protein synthesis in the retinas of C57BL/6J (n = 3), rd16 (n = 3), and rd16 4ebp1/2 −/− (n = 3) mice at P15. (D) ERG waveforms of three groups and (E) graph showing mean ERG a- and b- wave amplitudes of C57BL/6J (n = 4), rd16 (n = 6), and rd16 4ebp1/2 −/− (n = 6) mice at P17. (F) Representative images of H E-stained retinal sections taken at P18 in the regions denoted as * in (G). (G) Spider plots depicting number of photoreceptor nuclei in the ONL as counted by a masked investigator. The spidergram was generated by plotting the number of nuclei using 200 μm step in the distance from the ONH for both hemispheres. (H) TUNEL staining of retinal sections from C57BL/6J (n = 3), rd16 (n = 4), and rd16 4ebp1/2 −/− (n = 3) eyes at P15. (I) Graph showing TUNEL-positive nuclei per square millimeter in the three groups. Green, TUNEL; blue, DAPI; a.u, arbitrary units; kD, kilodaltons. Error bars: SEM. Statistical significance denoted by *P

    Article Snippet: Biotinylated proteins then were probed with HRP-conjugated streptavidin (S911; Thermo Fisher Scientific), incubated in ECL substrate (RPN2232; GE Healthcare, Chicago, IL, USA) and then imaged using a LI-COR (Lincoln, NE, USA) Fc imaging system.

    Techniques: Staining, Mouse Assay, Generated, TUNEL Assay