The ability of antibodies in immunized mouse sera to compete with both conformation-dependent and linear HCV E1E2-specific HMAbs was assessed by ELISA. The antibodies used for these experiments include AR3A and HEPC74 (domain B), HC84.26 and HC84.1 (domain D), HCV1 and HC33.1 (domain E), AR4A and AR5A (anti-E1E2), CBH-4G and CBH-4B (domain A), and H-111 and IGH526 (anti-E1). mbE1E2 was captured on GNA-coated microtiter plates at 4 °C for overnight. After blocking with Pierce Protein-Free Blocking Buffer (Thermo Fisher Scientific) for 1 h followed by three-time washing using Pierce Protein-Free Blocking Buffer, diluted mouse antisera (terminal bleed) were added to each well and incubated for 1 h at room temperature. After plates were washed with PBS containing 0.05% Tween 20, HCV E1E2-specific HMAbs were added at a concentration demonstrated previously to result in 70% of maximal binding and incubated for an additional hour. The HMAbs used for the competition ELISA were biotinylated using an EZ-Link NHS-PEO solid-phase biotinylation kit (Thermo Fisher Scientific). Bound biotinylated HMAb was detected using HRP-conjugated streptavidin (Abcam) at a dilution of 1:20,000. Absorbance was read at 450 nm using a SpectraMax M3 microplate reader. Percent inhibition values were calculated as the percentage of mAb binding relative to the mAb bound in the absence of serum.
Pierce protein free blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce Protein-Free Blocking Buffer is a blocking solution designed to reduce non-specific binding in immunoassays and Western blotting. It is a protein-free formulation that helps minimize interference from endogenous proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Maxisorp, by Thermo Fisher Scientific (4 mentions)
Galanthus nivalis lectin, by Vector Laboratories (4 mentions)
Tmb substrate, by Bio-Rad (3 mentions)
Tween 20, by Bio-Rad (2 mentions)
Igg2a, by Thermo Fisher Scientific (2 mentions)
Bright dab, by Immunologic (2 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Southern Biotech (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Ez link nhs peo solid phase biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Abcam (1 mentions)
Spectramax m3 microplate reader, by Molecular Devices (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
26 protocols using pierce protein free blocking buffer
1
HCV Antibody Competition ELISA
2
Quantifying E2-Specific Antibody Responses
3
Anti-α-Gal ELISA Protocol
4
Western Blot Analysis of Protein Modifications
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15 µg protein was incubated in 2x Laemmli buffer (Sigma) at 95 °C for 10 min and separated by a Mini-PROTEAN® TGX (Bio-Rad) precasted gel (4–20% acrylamide). Proteins were then transferred to a PVDF membrane and blocked with 2% dry milk (in PBS) or in the case of MG-modifications with a Pierce® protein-free blocking buffer (Thermo) at room temperature for 1 h. Membranes were then incubated overnight at 4 °C with antibodies against MG-H1 in protein-free blocking buffer (1:500 dilution), Actin in 2% dry milk containing PBS and 0.05% Tween20 (PBS-T) (1:1000 dilution) or GFP in PBS-T (1:1000 dilution). After 4 washing steps (10–15 min each) with PBS-T, membranes were incubated with secondary antibodies (anti-rat or anti-mouse, 1:2000 dilution) for 1 h at room temperature. Proteins were visualized on X-Ray films using ECL detection reagents (GE healthcare) with varying exposure time (0.5–5 min).
5
Quantifying sE2-specific Antibody Responses
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To detect sE2-specific total IgG, IgG1 and IgG2a responses in mouse serum, 96-well plates (MaxiSorp, Thermo Fisher, Waltham, MA) were coated overnight with 5 μg/mL Galanthus Nivalis Lectin (Vector Laboratories, Burlingame, CA, USA) at 4 °C. Plates were washed (PBS + 0.05% Tween 20) and coated with 200 ng/well E2 for 2 h at room temperature. After blocking with Pierce™ Protein-Free Blocking Buffer (Thermo Fisher, Waltham, MA, USA) for 1 h, serially diluted sera from mice were added to the plate and incubated for 2 h. The binding of sE2-specific antibodies was detected with 1:5000 dilutions of HRP-conjugated goat anti-mouse secondary antibodies to detect total IgG (H+L), IgG1 and IgG2a (Southern Biotech, Birmingham, AL, USA) with TMB substrate (Bio-Rad Laboratories, Hercules, CA, USA). Absorbance values at 450 nm (SpectraMax M3 microplate reader) were used to determine endpoint titers, which were calculated using curve fitting in GraphPad Prism software and defined as four times the highest absorbance value of pre-immune sera. Significance comparison was performed using a Kruskal–Wallis one-way ANOVA.
6
Western Blotting for Transcription Factor
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Western blotting for TF was performed as described previously [13 (link)]. Mouse recombinant TF was diluted in PBS. SDS sample buffer (1× final concentration, GenScript; Cat# M00676-250) and β-mercaptoethanol (2.5% final concentration) were added to rmTF. Samples were heated at 95 °C for 10 minutes and loaded on gels (4%-20% gradient, Mini-PROTEAN TGX gels, BioRad; Cat# 456-1093). SDS sample buffer and β-mercaptoethanol were added to the KPC2 Cas9 and KPC2 TF KO cell lysates, and 40 μg of protein were added per well. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (MilliporeSigma, Cat# IPFL00010) and the membrane was incubated with blocking buffer containing 5% nonfat dry milk in 1× tris-buffered saline, 0.1% Tween 20 detergent (TBS-T buffer) for 1 hour at room temperature. The membrane was incubated with a rabbit anti-mouse TF antibody (Abcam; Cat# ab189483, 1:1000) in Pierce Protein-Free Blocking Buffer (Thermo Fisher Scientific, Cat# 37572) overnight at 4 °C, washed, and then incubated with a goat anti-rabbit IgG-HRP (Cell Signaling; Cat# 7074, 1:5000). Membranes were developed using chemiluminescence (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific; Cat# 34577) and detected using a ChemiDoc MP Imaging System (BioRad).
7
Ebola Glycoprotein Antibody ELISA
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EBOV GP-specific antibody responses were evaluated by antigen-capture enzyme-linked immunosorbent assay (ELISA) using the immune sera as previously described [49 (link)]. In brief, 96-well plates (MaxiSorp, ThermoFisher Scientific) were coated with 5 µg/mL Galanthus Nivalis Lectin (Vector Laboratories, Burlingame, CA, USA) overnight at 4 °C. The following day, plates were washed with PBS containing 0.05% Tween 20 and then coated with 200 ng/well Zaire GPΔmuc antigen at 4 °C. Plates were washed 3 times after overnight incubation and blocked with Pierce Protein-Free Blocking Buffer (ThermoFisher Scientific) for 1 h at room temperature. Serum from individual mice was then added to the plates and tested in duplicate at 5-fold serial dilutions. The binding of Zaire GP-specific antibodies was detected by 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Abcam, Cambridge, MA, USA), followed by incubation with 100 µL of TMB substrate (Bio-Rad Laboratories, Hercules, CA, USA) for color development. The absorbance was measured at 450 nm using SpectraMax M3 microplate reader (Molecular Devices, San Jose, CA, USA). The antibody endpoint titer was determined as the highest reciprocal dilution of serum that resulted an optical density (OD) reading 4 times the value of pre-immune sera.
8
Quantitative Western Blot Analysis
9
Western Blot Analysis of GSCs
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RIPA buffer with 0.01% of a protease and phosphatase inhibitor cocktail was used to lyse cells to prepare GSCs. A bicinchoninic acid protein assay was used to calculate protein concentrations. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 4%–12% gradient gel was used to separate 40 μg samples of denatured proteins. After transfer to polyvinylidene fluoride membranes, these were blocked in Pierce Protein Free blocking buffer (Thermo Fisher Scientific). Membranes were incubated overnight at 4°C with primary antibodies. Anti-GAPDH antibody was used to check for equal loading. Secondary antibodies used were horse radish peroxidase goat anti-mouse IgG or anti-rabbit IgG, and an ECL kit used to visualize immunoreactive protein bands.
10
Fbs1 Affinity Purification Protocol
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One millilitre of the E. coli cell lysate containing wt Fbs1 or Fbs1 mutant proteins was incubated with 50 μl fetuin or RNase B beads at 4 °C for 1 h in optimal binding buffers (20 mM Tris.HCl pH 7.5, 1 mM EDTA, 50 mM NaCl (for low-salt conditions) or 2 M NaCl (for high-salt conditions)) supplemented with 30% (v/v) of Pierce Protein-Free Blocking buffer (Thermo Fisher Scientific). After incubation, the beads were washed three times with 1 ml of the corresponding binding buffers. In each wash, 30-gauge insulin syringes (Becton Dickinson, Franklin Lakes, NJ) were used to remove residual buffer. SDS–PAGE loading buffer was used to elute Fbs1 bound to the beads.
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