Structured Review

Agilent technologies goat igg ab
Goat Igg Ab, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/goat igg ab/product/Agilent technologies
Average 85 stars, based on 2 article reviews
Price from $9.99 to $1999.99
goat igg ab - by Bioz Stars, 2020-09
85/100 stars

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Staining:

Article Title: Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis
Article Snippet: .. Then the tissue sections were subjected to the second staining for NFAT2 with the sequential steps of quenching the endogenous phosphatase, incubation with anti-NFAT2 antibodies, incubation with anti-goat IgG antibodies and detection of the phosphatase activity in accordance with the manufacturer’s instructions (Envision Doublestain System, DAKO, Glostrup, Denmark). .. For confocal microscopy, tissue sections were de-waxed using xylene twice and rehydrated with PBS followed by blocking with goat serum (BioGenex, San Ramon, CA 94583 USA) for 10 min at room temperature.

Incubation:

Article Title: The Anti-Tumor Effects and Molecular Mechanisms of Suberoylanilide Hydroxamic Acid (SAHA) on the Aggressive Phenotypes of Ovarian Carcinoma Cells
Article Snippet: .. For immunoblotting, the membranes were incubated for 1 h with the primary antibody (Table S2 in ), rinsed with TBST, and incubated with anti-rabbit, anti-mouse or anti-goat IgG antibodies conjugated to horseradish peroxidase (HRP; Dako, Carpinteria CA, USA) at a dilution of 1:1000. .. After applying electrochemiluminescent (ECL)-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the protein bands were visualized using X-ray film (Fujifilm, Tokyo, Japan).

Article Title: Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis
Article Snippet: .. Then the tissue sections were subjected to the second staining for NFAT2 with the sequential steps of quenching the endogenous phosphatase, incubation with anti-NFAT2 antibodies, incubation with anti-goat IgG antibodies and detection of the phosphatase activity in accordance with the manufacturer’s instructions (Envision Doublestain System, DAKO, Glostrup, Denmark). .. For confocal microscopy, tissue sections were de-waxed using xylene twice and rehydrated with PBS followed by blocking with goat serum (BioGenex, San Ramon, CA 94583 USA) for 10 min at room temperature.

Article Title: The Involvement of RhoA and Wnt-5a in the Tumorigenesis and Progression of Ovarian Epithelial Carcinoma
Article Snippet: .. Then, the membranes were rinsed with TBST and incubated with anti-mouse, anti-rabbit, or anti-goat IgG antibodies conjugated to horseradish peroxidase (1:1000; Dako, Carpinteria, CA, USA) for 15 min. All incubations were performed in a microwave oven (Oriental Rotor, Tokyo, Japan) with intermittent irradiation. .. Bands were visualized on X-ray film (Fujifilm, Tokyo, Japan) using ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan) and ECL Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Article Title: SAHA and/or MG132 reverse the aggressive phenotypes of glioma cells: An in vitro and vivo study
Article Snippet: .. The membranes were rinsed with TBST, and incubated with anti-rabbit, anti-mouse or anti-goat IgG antibody conjugated to horseradish peroxidase (HRP, Dako, USA). .. Bands were visualized with LAS4010 (GE healthcare Life Science, USA) by ECL-Plus detection reagents (Santa Cruz, USA).

Article Title: Anacardic Acid Enhances the Proliferation of Human Ovarian Cancer Cells
Article Snippet: .. For immunoblotting, the membranes were incubated for 1 h with the primary antibody, rinsed with TBST and incubated with anti-rabbit, anti-mouse or anti-goat IgG antibodies conjugated to horseradish peroxidase (HRP; Dako, Carpinteria CA, USA) at a dilution of 1∶1000. .. After applying enhanced chemiluminescent (ECL)-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the protein bands were visualized using an X-ray film (Fujifilm, Tokyo, Japan).

other:

Article Title: Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model
Article Snippet: Rabbit anti-AQP2 polyclonal antibody (catalog no. AQP-002) was from Alomone Labs (Jerusalem, Israel), rabbit anti-TSG101 monoclonal antibody (catalog no. ab125011) was from Abcam (Cambridge, UK), goat anti-ALIX polyclonal antibody (catalog no. sc49268) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), peroxidase-conjugated anti-rabbit IgG antibody (catalog no. 7074) was from Cell Signaling Technology (Danvers, MA, USA), and anti-goat IgG antibody (catalog no. P0449) was from Dako Japan (Tokyo, Japan), were used.

Activity Assay:

Article Title: Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis
Article Snippet: .. Then the tissue sections were subjected to the second staining for NFAT2 with the sequential steps of quenching the endogenous phosphatase, incubation with anti-NFAT2 antibodies, incubation with anti-goat IgG antibodies and detection of the phosphatase activity in accordance with the manufacturer’s instructions (Envision Doublestain System, DAKO, Glostrup, Denmark). .. For confocal microscopy, tissue sections were de-waxed using xylene twice and rehydrated with PBS followed by blocking with goat serum (BioGenex, San Ramon, CA 94583 USA) for 10 min at room temperature.

Irradiation:

Article Title: The Involvement of RhoA and Wnt-5a in the Tumorigenesis and Progression of Ovarian Epithelial Carcinoma
Article Snippet: .. Then, the membranes were rinsed with TBST and incubated with anti-mouse, anti-rabbit, or anti-goat IgG antibodies conjugated to horseradish peroxidase (1:1000; Dako, Carpinteria, CA, USA) for 15 min. All incubations were performed in a microwave oven (Oriental Rotor, Tokyo, Japan) with intermittent irradiation. .. Bands were visualized on X-ray film (Fujifilm, Tokyo, Japan) using ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan) and ECL Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

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  • 92
    Agilent technologies goat anti mouse igg hrp
    Optimisation of culture conditions and r CLU expression in MEXi 293E cells. ( A ) and ( B ) Data shown for MEXi293E cells transfected with pEGFP-N1; means + /− SEM (n = 3) are plotted. In some cases the error bars are too small to be visible. ( A ) Left Panel: Viable cell density over 5 days post transfection (DPT). Right Panel: Percent cell viability over 5 DPT. ( B ) Percent of cells transiently expressing GFP measured for 5 DPT. ( C ) Transfection efficiency of cells overexpressing r CLU-αC-ST and r CLU-CT were measured by immunostaining of fixed, permeabilised cells on day 5 post-transfection. Untransfected (UN) cells were used as control. To detect r CLU-αC-ST, cells were stained with anti-strep tag antibody (Anti-ST Ab) or DNP9 (an isotype-matched antibody of irrelevant specificity) followed by goat anti-mouse <t>IgG-CF488.</t> r CLU-CT was detected using biotinylated anti-C tag conjugate ( b -CT-Ab) followed by streptavidin-CF488 (SA-488); the negative control in this case was cells incubated with SA-488 alone. ( D ) Densitometric analysis of Western blot with culture supernatant from cells overexpressing r CLU-αC-ST harvested on day 5 DPT and probed with <t>streptactin-HRP</t> conjugate. Cells were either supplemented with tryptone N1 (TN1) or sodium butyrate (NaB) or untreated (UN). Means + /− SEM (n = 3) are plotted; statistically significant differences are indicated by * (Oneway ANOVA, p
    Goat Anti Mouse Igg Hrp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igg hrp/product/Agilent technologies
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igg hrp - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    85
    Agilent technologies horseradish peroxidase hrp conjugated goat anti mouse immunoglobulin g igg
    Expression of HIF-1α in TB granulomas and macrophages. (A) The lungs of each of seven BALB/c mice 5 weeks after infection with or without M. tuberculosis Erdman were fixed in 10% formaldehyde. A normal mouse lung is shown in i–iv and mouse lung granuloma in v–viii. The mouse lung sections were stained with Hematoxylin-Eosin stain (i, v) or the Ziehl–Neelsen stain (ii, vi), and immune-stained with anti-HIF-1α <t>IgG</t> (iii, vii) and anti-MDP1 IgG (iv, viii). Scale bars: 100 μm (i–viii). (B and C) After infection with M. tuberculosis H37Rv (Mtb) or M. bovis BCG (BCG) for 12 h, BMDMs from WT mice were cultured for 24 h in the absence or presence of 500 U ml −1 IFN-γ. (B) Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-HIF-1α antibody, anti-HIF-1β antibody or anti-β-actin antibody (as the loading control). Ratios of HIF-1α/β-actin are shown as relative intensities. Values are expressed as means ± SD from four independent biological replicates. (C) Amplified Hif-1α mRNA products were normalized to 18S rRNA. Values are expressed as the means ± SD from three independent biological replicates. (D) After infection with M. tuberculosis H37Rv for 12 h, BMDMs from WT mice or those lacking Toll-like receptor 2/4/9 (TLR2/4/9) were cultured for 24 h without 500 U ml −1 IFN-γ. Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with SDS–PAGE and immunoblotted with an anti-HIF-1α antibody or anti-β-actin antibody (as the loading control). The significance of differences was assessed by a two-way analysis of variance (ANOVA) followed by the Bonferroni test.
    Horseradish Peroxidase Hrp Conjugated Goat Anti Mouse Immunoglobulin G Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase hrp conjugated goat anti mouse immunoglobulin g igg/product/Agilent technologies
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase hrp conjugated goat anti mouse immunoglobulin g igg - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    91
    Agilent technologies hrp conjugated rabbit anti goat igg
    Ionic strength–dependent interaction of CIP, with C3d assessed by ELISA. Microtiter wells were coated with 250 ng of C3d. The wells were probed with CIP or CD21 diluted in a buffer containing increasing concentrations of NaCl (0–2.5 M). Complex formation was revealed through anti-CIP or anti-CD21 <t>IgG</t> pAb, followed by an <t>HRP-conjugated</t> secondary antibody.
    Hrp Conjugated Rabbit Anti Goat Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated rabbit anti goat igg/product/Agilent technologies
    Average 91 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated rabbit anti goat igg - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Optimisation of culture conditions and r CLU expression in MEXi 293E cells. ( A ) and ( B ) Data shown for MEXi293E cells transfected with pEGFP-N1; means + /− SEM (n = 3) are plotted. In some cases the error bars are too small to be visible. ( A ) Left Panel: Viable cell density over 5 days post transfection (DPT). Right Panel: Percent cell viability over 5 DPT. ( B ) Percent of cells transiently expressing GFP measured for 5 DPT. ( C ) Transfection efficiency of cells overexpressing r CLU-αC-ST and r CLU-CT were measured by immunostaining of fixed, permeabilised cells on day 5 post-transfection. Untransfected (UN) cells were used as control. To detect r CLU-αC-ST, cells were stained with anti-strep tag antibody (Anti-ST Ab) or DNP9 (an isotype-matched antibody of irrelevant specificity) followed by goat anti-mouse IgG-CF488. r CLU-CT was detected using biotinylated anti-C tag conjugate ( b -CT-Ab) followed by streptavidin-CF488 (SA-488); the negative control in this case was cells incubated with SA-488 alone. ( D ) Densitometric analysis of Western blot with culture supernatant from cells overexpressing r CLU-αC-ST harvested on day 5 DPT and probed with streptactin-HRP conjugate. Cells were either supplemented with tryptone N1 (TN1) or sodium butyrate (NaB) or untreated (UN). Means + /− SEM (n = 3) are plotted; statistically significant differences are indicated by * (Oneway ANOVA, p

    Journal: Scientific Reports

    Article Title: Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants

    doi: 10.1038/s41598-020-70990-3

    Figure Lengend Snippet: Optimisation of culture conditions and r CLU expression in MEXi 293E cells. ( A ) and ( B ) Data shown for MEXi293E cells transfected with pEGFP-N1; means + /− SEM (n = 3) are plotted. In some cases the error bars are too small to be visible. ( A ) Left Panel: Viable cell density over 5 days post transfection (DPT). Right Panel: Percent cell viability over 5 DPT. ( B ) Percent of cells transiently expressing GFP measured for 5 DPT. ( C ) Transfection efficiency of cells overexpressing r CLU-αC-ST and r CLU-CT were measured by immunostaining of fixed, permeabilised cells on day 5 post-transfection. Untransfected (UN) cells were used as control. To detect r CLU-αC-ST, cells were stained with anti-strep tag antibody (Anti-ST Ab) or DNP9 (an isotype-matched antibody of irrelevant specificity) followed by goat anti-mouse IgG-CF488. r CLU-CT was detected using biotinylated anti-C tag conjugate ( b -CT-Ab) followed by streptavidin-CF488 (SA-488); the negative control in this case was cells incubated with SA-488 alone. ( D ) Densitometric analysis of Western blot with culture supernatant from cells overexpressing r CLU-αC-ST harvested on day 5 DPT and probed with streptactin-HRP conjugate. Cells were either supplemented with tryptone N1 (TN1) or sodium butyrate (NaB) or untreated (UN). Means + /− SEM (n = 3) are plotted; statistically significant differences are indicated by * (Oneway ANOVA, p

    Article Snippet: Bound primary antibodies were detected using goat anti-mouse IgG-HRP (Dako Agilent) diluted 1:5,000 in SM/PBS.

    Techniques: Expressing, Transfection, Immunostaining, Staining, Strep-tag, Negative Control, Incubation, Western Blot

    Expression of HIF-1α in TB granulomas and macrophages. (A) The lungs of each of seven BALB/c mice 5 weeks after infection with or without M. tuberculosis Erdman were fixed in 10% formaldehyde. A normal mouse lung is shown in i–iv and mouse lung granuloma in v–viii. The mouse lung sections were stained with Hematoxylin-Eosin stain (i, v) or the Ziehl–Neelsen stain (ii, vi), and immune-stained with anti-HIF-1α IgG (iii, vii) and anti-MDP1 IgG (iv, viii). Scale bars: 100 μm (i–viii). (B and C) After infection with M. tuberculosis H37Rv (Mtb) or M. bovis BCG (BCG) for 12 h, BMDMs from WT mice were cultured for 24 h in the absence or presence of 500 U ml −1 IFN-γ. (B) Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-HIF-1α antibody, anti-HIF-1β antibody or anti-β-actin antibody (as the loading control). Ratios of HIF-1α/β-actin are shown as relative intensities. Values are expressed as means ± SD from four independent biological replicates. (C) Amplified Hif-1α mRNA products were normalized to 18S rRNA. Values are expressed as the means ± SD from three independent biological replicates. (D) After infection with M. tuberculosis H37Rv for 12 h, BMDMs from WT mice or those lacking Toll-like receptor 2/4/9 (TLR2/4/9) were cultured for 24 h without 500 U ml −1 IFN-γ. Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with SDS–PAGE and immunoblotted with an anti-HIF-1α antibody or anti-β-actin antibody (as the loading control). The significance of differences was assessed by a two-way analysis of variance (ANOVA) followed by the Bonferroni test.

    Journal: International Immunology

    Article Title: Metabolic adaptation to glycolysis is a basic defense mechanism of macrophages for Mycobacterium tuberculosis infection

    doi: 10.1093/intimm/dxz048

    Figure Lengend Snippet: Expression of HIF-1α in TB granulomas and macrophages. (A) The lungs of each of seven BALB/c mice 5 weeks after infection with or without M. tuberculosis Erdman were fixed in 10% formaldehyde. A normal mouse lung is shown in i–iv and mouse lung granuloma in v–viii. The mouse lung sections were stained with Hematoxylin-Eosin stain (i, v) or the Ziehl–Neelsen stain (ii, vi), and immune-stained with anti-HIF-1α IgG (iii, vii) and anti-MDP1 IgG (iv, viii). Scale bars: 100 μm (i–viii). (B and C) After infection with M. tuberculosis H37Rv (Mtb) or M. bovis BCG (BCG) for 12 h, BMDMs from WT mice were cultured for 24 h in the absence or presence of 500 U ml −1 IFN-γ. (B) Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an anti-HIF-1α antibody, anti-HIF-1β antibody or anti-β-actin antibody (as the loading control). Ratios of HIF-1α/β-actin are shown as relative intensities. Values are expressed as means ± SD from four independent biological replicates. (C) Amplified Hif-1α mRNA products were normalized to 18S rRNA. Values are expressed as the means ± SD from three independent biological replicates. (D) After infection with M. tuberculosis H37Rv for 12 h, BMDMs from WT mice or those lacking Toll-like receptor 2/4/9 (TLR2/4/9) were cultured for 24 h without 500 U ml −1 IFN-γ. Whole-cell lysates were fractionated on 7.5% polyacrylamide gels with SDS–PAGE and immunoblotted with an anti-HIF-1α antibody or anti-β-actin antibody (as the loading control). The significance of differences was assessed by a two-way analysis of variance (ANOVA) followed by the Bonferroni test.

    Article Snippet: Materials EnVision™ kits, horseradish-peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG were obtained from Dako/Agilent (Santa Clara, CA, USA).

    Techniques: Expressing, Mouse Assay, Infection, Staining, Ziehl-Neelsen Stain, Cell Culture, Polyacrylamide Gel Electrophoresis, SDS Page, Amplification

    Ionic strength–dependent interaction of CIP, with C3d assessed by ELISA. Microtiter wells were coated with 250 ng of C3d. The wells were probed with CIP or CD21 diluted in a buffer containing increasing concentrations of NaCl (0–2.5 M). Complex formation was revealed through anti-CIP or anti-CD21 IgG pAb, followed by an HRP-conjugated secondary antibody.

    Journal: The FASEB Journal

    Article Title: The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands

    doi: 10.1096/fj.201801991R

    Figure Lengend Snippet: Ionic strength–dependent interaction of CIP, with C3d assessed by ELISA. Microtiter wells were coated with 250 ng of C3d. The wells were probed with CIP or CD21 diluted in a buffer containing increasing concentrations of NaCl (0–2.5 M). Complex formation was revealed through anti-CIP or anti-CD21 IgG pAb, followed by an HRP-conjugated secondary antibody.

    Article Snippet: The wells were washed 3 times with PBST, blocked with 2% (w/v) BSA in PBST for 1 h at 22°C, and then probed with C3d (100 ng/well) preincubated with serial dilutions of CIP in PBS, followed by incubation with anti-C3 goat pAb (1:2000; Complement Technology) and then HRP-conjugated rabbit anti-goat IgG (1:1000; Agilent Technologies).

    Techniques: Enzyme-linked Immunosorbent Assay

    Competitive inhibition assessment of CIP interaction with C3b and C4b. A ) Results of an experiment with 100 ng of C3b was coated on a 96-well plate and overlaid with 0.6 µM of CIP preincubated with equimolar amounts of C3b or C4b, followed by incubation with anti-CIP IgG pAb and then an anti-mouse HRP-conjugated antibody. B ) A similar experiment in which 100 ng of C4b was immobilized and overlaid with 0.6 µM of CIP preincubated with equimolar amounts of C4b or C3b.

    Journal: The FASEB Journal

    Article Title: The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands

    doi: 10.1096/fj.201801991R

    Figure Lengend Snippet: Competitive inhibition assessment of CIP interaction with C3b and C4b. A ) Results of an experiment with 100 ng of C3b was coated on a 96-well plate and overlaid with 0.6 µM of CIP preincubated with equimolar amounts of C3b or C4b, followed by incubation with anti-CIP IgG pAb and then an anti-mouse HRP-conjugated antibody. B ) A similar experiment in which 100 ng of C4b was immobilized and overlaid with 0.6 µM of CIP preincubated with equimolar amounts of C4b or C3b.

    Article Snippet: The wells were washed 3 times with PBST, blocked with 2% (w/v) BSA in PBST for 1 h at 22°C, and then probed with C3d (100 ng/well) preincubated with serial dilutions of CIP in PBS, followed by incubation with anti-C3 goat pAb (1:2000; Complement Technology) and then HRP-conjugated rabbit anti-goat IgG (1:1000; Agilent Technologies).

    Techniques: Inhibition, Incubation

    Comparison of micro-refold versus UV exchange peptide binding assay approaches A. Average absorbance readings for 2 HLA-E binding pathogen-derived peptides sampled in (i) the previously published micro-refolding peptide binding affinity assay, (ii) the original (standard) UV-exchange peptide binding affinity approach and (iii) the optimised UV exchange peptide binding assay approach. The Y-axis denotes average absorbance readings at 450nm and the X-axis denotes test peptides, which are colour-coded according to the shared Figure legend. Peptide to background ratios are indicated above each bar, with peptide ID and sequences denoted. The sequence origin and corresponding references are detailed in Supplementary Table 1, C. The mouse anti-human HLA-E monoclonal antibody 3D12 was used as the ELISA capture antibody, with polyclonal rabbit anti-human β2M HRP-conjugated IgG antibody used as detection, followed by goat-anti-rabbit EnVision+ System-HRP signal amplification. The positive control peptide VL9 (HLA-B7-derived leader sequence peptide, VMAPRTVLL) is shown in dark blue. Peptide free negative background are noted in grey; for micro-refold approach this consisted of HLA-E heavy chain and β2M only (HC β2M) whereas the UV peptide exchange approach negative control comprised previously refolded HLA-E complexes with no added rescue peptide in the exchange reaction (No Rescue). Three biological repeats (three individual peptide exchange reactions per test peptide) were carried out simultaneously, with two technical replicas sampled from the same peptide exchange reaction performed for each biological repeat. (reported herein as n=3, technical replica n=2). Stars above error bars reflect degree of significance in peptide binding by two-tailed t-tests: no star=P > 0.05, *=P≤0.05, **=P≤0.01, ***=P≤0.001, ****P=≤0.0001. Error bars depicting the mean and standard deviation of biological repeats are shown. The conditions outlined in italics are replicated in all subsequent ELISA optimisation assays. B. Table of standard deviation for the micro-refold and the standard and optimised UV exchange peptide binding assay approaches.

    Journal: bioRxiv

    Article Title: Detailed and atypical HLA-E peptide binding motifs revealed by a novel peptide exchange binding assay

    doi: 10.1101/2020.05.05.078790

    Figure Lengend Snippet: Comparison of micro-refold versus UV exchange peptide binding assay approaches A. Average absorbance readings for 2 HLA-E binding pathogen-derived peptides sampled in (i) the previously published micro-refolding peptide binding affinity assay, (ii) the original (standard) UV-exchange peptide binding affinity approach and (iii) the optimised UV exchange peptide binding assay approach. The Y-axis denotes average absorbance readings at 450nm and the X-axis denotes test peptides, which are colour-coded according to the shared Figure legend. Peptide to background ratios are indicated above each bar, with peptide ID and sequences denoted. The sequence origin and corresponding references are detailed in Supplementary Table 1, C. The mouse anti-human HLA-E monoclonal antibody 3D12 was used as the ELISA capture antibody, with polyclonal rabbit anti-human β2M HRP-conjugated IgG antibody used as detection, followed by goat-anti-rabbit EnVision+ System-HRP signal amplification. The positive control peptide VL9 (HLA-B7-derived leader sequence peptide, VMAPRTVLL) is shown in dark blue. Peptide free negative background are noted in grey; for micro-refold approach this consisted of HLA-E heavy chain and β2M only (HC β2M) whereas the UV peptide exchange approach negative control comprised previously refolded HLA-E complexes with no added rescue peptide in the exchange reaction (No Rescue). Three biological repeats (three individual peptide exchange reactions per test peptide) were carried out simultaneously, with two technical replicas sampled from the same peptide exchange reaction performed for each biological repeat. (reported herein as n=3, technical replica n=2). Stars above error bars reflect degree of significance in peptide binding by two-tailed t-tests: no star=P > 0.05, *=P≤0.05, **=P≤0.01, ***=P≤0.001, ****P=≤0.0001. Error bars depicting the mean and standard deviation of biological repeats are shown. The conditions outlined in italics are replicated in all subsequent ELISA optimisation assays. B. Table of standard deviation for the micro-refold and the standard and optimised UV exchange peptide binding assay approaches.

    Article Snippet: After 30mins of dark incubation at 4°C, ELISA plates were washed and 50µL of a goat anti-rabbit HRP-conjugated IgG enhancement antibody (EnVision+ System-HRP from Agilent), diluted 1:15 in 2% BSA containing 1% mouse serum to prevent cross-reactivity, was added to each well to amplify the signal.

    Techniques: Binding Assay, Derivative Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Amplification, Positive Control, Negative Control, Two Tailed Test, Standard Deviation