Measurements of binding antibody levels to various SARS-CoV-2 strains were performed on sera from immunized mice, as previously described [22 (link)]. Briefly, ELISA plates were coated with 0.5 µg/ml of S protein purchased from ACRO Biosystems (Alpha strain, SPN-C52H6; Delta strain, SPN-C52He; Omicron BA.5, SPN-C522e) and incubated at 4 °C overnight, followed by blocking with PBS containing 1% bovine serum albumin (BSA) at RT for 2 h. After blocking, the wells were washed twice with PBS. Then, sera that were serially diluted with 1% BSA in PBS were added into each well in triplicate, and the plate was incubated at room temperature for 1 h. After an incubation period, the plates were washed three times with PBS containing 0.1% Tween-20 (PBST0.1) and then incubated for 1 h with peroxidase-affinipure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch) (1:5000 dilution). After three washes with PBST0.1, signal was produced using 3,3’5,5’-Tetramethylbenzidine (TMB) substrate (TMBW-1000-01, SURMODICS). Finally, the reaction was stopped with 3 N HCl, and absorbance was measured at 450 nm by an ELISA reader (Versa Max Tunable Microplate Reader; Molecular Devices).
Peroxidase affinipure goat anti mouse igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom, Panama
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) is a secondary antibody produced in goat and purified through affinity chromatography. It is conjugated with horseradish peroxidase (HRP) and specifically binds to the heavy and light chains of mouse immunoglobulin G (IgG).
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Lab products found in correlation
Peroxidase affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (19 mentions)
Versamax tunable microplate reader, by Molecular Devices (2 mentions)
Goat anti rabbit igg h l, by Jackson ImmunoResearch (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Capture anti mouse ifn γ mab, by BioLegend (2 mentions)
Peroxidase conjugated affinipure donkey anti mouse lgm, by Jackson ImmunoResearch (2 mentions)
Anti mouse cd 3 antibody, by BioXCell (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Peroxidase affinipure goat anti rat igg h l, by Jackson ImmunoResearch (2 mentions)
Affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (2 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Visionworks ls software, by Analytik Jena (2 mentions)
Pvdf membrane, by Roche (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Las 4010 imaging system, by GE Healthcare (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
42 protocols using peroxidase affinipure goat anti mouse igg h l
1
SARS-CoV-2 Antibody Binding Assay
2
Western Blot Analysis of Protein Expression
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Whole-cell extracts were prepared by incubating cells in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche, Penzberg, Germany) for 30 min on ice. The supernatant was collected by centrifuge and protein concentration was quantified using the Pierce BCA Kit (Thermo Fisher Scientific). Blotting was conducted as previously described (33 (link)). Primary antibodies against the following proteins were used: BRD2 (Cat: 5848s, 1:1,000, Cell Signaling Technology), BRD3 (Cat: 11859-1-AP, 1:1,000, Proteintech), BRD4 (Cat: 13440s, 1:1000, Cell Signaling Technology), CRBN (Cat: HPA045910, Sigma-Aldrich), c-Myc (Cat: 9402, 1:1,000, Cell Signaling Technology), MYCN (Cat: sc-53993, 1:1,000, Santa Cruz Biotechnology), cleaved-Caspase 3 (Cat: 9664, 1:1,000, Cell Signaling Technology), PARP (Cat: 9542, 1:1,000, Cell Signaling Technology). β-actin (Cat: A5441, 1:5,000, Sigma-Aldrich) or GAPDH (1:2,000; MA3374, Millipore) were used as a reference protein. The horseradish peroxidase-conjugated secondary antibodies Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L) (Cat: 115-035-003) and Goat Anti-Rabbit IgG(H+L) (Cat: 111-035-003) were purchased from Jackson ImmunoResearch Laboratories, INC. The bands were visualized by an ECL detection kit (Pierce, Rockford, IL, USA) using LAS 4010 imaging system (GE Healthcare Life Sciences, Little Chalfont, UK).
3
Comprehensive Western Blot Analysis Protocol
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Western blot was performed as previously described (11 (link)). The primary antibodies used included anti-CD206 (ab64693, Abcam), anti-Arg-1 (ab233548, Abcam), anti-Ym1 (ab192029, Abcam), anti-SIRPα (ab191419, Abcam), anti-p-STAT3 S727 (ab32143, Abcam), anti-p-STAT3 Y705 (#9664, CST), anti-STAT3 (9662, CST), anti-α-SMA (ab7817, Abcam), anti-cleaved Caspase 3 (CST; 9662), anti-Caspase 3 (CST; #3498), anti-Bcl2 (CST; #3498), anti-E-cadherin (GTX100443, GTX), and anti-GAPDH (ab181602, Abcam). The secondary antibodies included Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (111035003, 115035003, Jackson, West Grove, PA, USA). Protein levels were quantified using the Image Lab software, version 3.0 (Bio-Rad, Hercules, CA, USA).
4
Halo-Tagged Protein Detection in Virus-Infected Cells
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Virus-infected Min6 cells were lysed with sample buffer as described above, and separated using a Tris-Tricine SDS–PAGE as described27 . The lysed islets samples were resolved using a 16% polyacrylamide gel, blotted to a PVDF membrane (Millipore, Germany), and probed overnight at 4 °C with antibodies against Halo-Tag (Promega). The secondary antibody used was Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson). Detections were performed using FUSION-SOLO.4S.WL (M&S instruments, Tokyo, Japan).
5
Protein Extraction and Western Blot Analysis
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Cellular proteins were extracted using the following lysis buffer: 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 10% glycerol, 1% NP-40, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM N-ethylmaleimide, and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Lysis was performed on ice with agitation for 30 minutes, followed by 30-minute centrifugation for 30 minutes at 16,000g at 4°C. Clarified lysates were then denatured at 95°C for 8 minutes in the presence of 4× LDS sample loading buffer (Invitrogen) and 5% β-mercaptoethanol (Bio-Rad), and they were separated by SDS-PAGE with NuPAGE 4%–12% Bis-Tris 1.5 mm 15-well gels (Thermo Fisher Scientific). Proteins were then transferred to nitrocellulose using iBlot2 system (Thermo Fisher Scientific), and membranes were blocked in 5% milk/TBS-T for 1 hour, followed by incubation with primary antibody overnight. After washes and incubation with respective horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Peroxidase AffiniPure goat anti–mouse IgG (H+L) [catalog 115-035-146] at 1:10,000 or Peroxidase AffiniPure goat anti–rabbit IgG (H+L) [catalog 111-035-003] at 1:20,000) for 1 hour, protein bands were visualized using SuperSignal West Femto substrate (Thermo Fisher Scientific) with Image-Quant LAS 4000 biomolecular imager (GE Healthcare Life Sciences).
6
Quantifying Antibody Binding to Viral Proteins
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The ELISA plates were coated with 0.5 μg/ml RBD-His, S-His, or EpEX-His protein in 0.1 M NaHCO3 (pH 8.6) buffer at 4°C overnight, followed by blocking with PBS containing 1% bovine serum albumin (BSA) at RT for 2 h. After blocking, the wells were washed twice with PBS; the plates were then stored at -20°C.
The protein contents of the culture supernatants from hybridoma or antibodies were quantified by the BCA assay and serially diluted with 1% BSA in PBS. Then, 50 μl supernatant or antibody was added into each well, and the plate was incubated for 1 h at room temperature. The plates were washed with PBS containing 0.1% Tween-20 (PBST0.1) three times and then incubated for 1 h with Peroxidase AffiniPure Goat Anti-mouse IgG (H+L) (Jackson ImmunoResearch) or Peroxidase AffiniPure Goat Anti-human IgG (H+L) (Jackson ImmunoResearch) (1:5000 dilution), as appropriate. After three washes with PBST0.1, signal was produced using 3,3’5,5’-Tetramethylbenzidine (TMB) color development (TMBW-1000-01, SURMODICS). The reaction was stopped with 3 N HCl, and absorbance was measured at 450 nm by ELISA reader (Versa Max Tunable Microplate Reader; Molecular Devices).
The protein contents of the culture supernatants from hybridoma or antibodies were quantified by the BCA assay and serially diluted with 1% BSA in PBS. Then, 50 μl supernatant or antibody was added into each well, and the plate was incubated for 1 h at room temperature. The plates were washed with PBS containing 0.1% Tween-20 (PBST0.1) three times and then incubated for 1 h with Peroxidase AffiniPure Goat Anti-mouse IgG (H+L) (Jackson ImmunoResearch) or Peroxidase AffiniPure Goat Anti-human IgG (H+L) (Jackson ImmunoResearch) (1:5000 dilution), as appropriate. After three washes with PBST0.1, signal was produced using 3,3’5,5’-Tetramethylbenzidine (TMB) color development (TMBW-1000-01, SURMODICS). The reaction was stopped with 3 N HCl, and absorbance was measured at 450 nm by ELISA reader (Versa Max Tunable Microplate Reader; Molecular Devices).
7
Recombinant ACE2 and VE-cadherin Protein Analysis
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Recombinant Human ACE2 Protein (933-ZN) was purchased from R&D Systems (Minneapolis, MN, USA) and stored in a buffer according to the manufacturer’s instructions. Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant VE-cadherin extracellular domain (EC1-5) was produced in the laboratory. Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338447), Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (AB-2307391), and Cy3™ AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338680) were all purchased from Jackson Immunoresearch (Ely, Cambridgeshire, UK). Hoechst solution (33,258) was purchased from Sigma Aldrich. The mouse monoclonal anti-human VE-cadherin antibody (clone BV9) and the rabbit polyclonal anti-EC1 anti-human VE-cadherin antibody were produced in the laboratory.
8
Enzyme-Linked Immunosorbent Assay for Antibody Detection
9
Antibody-Based Cytokine Detection Assay
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Capture anti-mouse IFN-γ mAb (clone: R4–6A2) and biotinylated, detection anti-mouse IFN-γ mAb (clone: XMG1.2-Biotin) were purchased from BioLegend, San Diego, CA. Peroxidase-conjugated AffiniPure Donkey Anti-Mouse lgM and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) were purchased from Jackson ImmunoResearch Inc. Anti-mouse CD-3 antibody (clone: 145–2C11) was purchased from BioXCell, West Lebanon, NH. FITC-, PE-, APC- or biotin-conjugated monoclonal antibodies to Foxp3 (clone: MF-14), B220 (clone: RA3–6B2), CD4 (clone: GK1.5), and CD8 (clone: 53–6.7) were purchased from BioLegend, San Diego, CA.
10
Protein Extraction and Western Blot
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Cells were collected by trypsinization and washed 3 times with PBS (1,000 × g, 5 minutes, 4°C). Cell pellets were resuspended in lysis buffer (1% (w/v) digitonin, 1× Roche cOmplete protease inhibitor, 0.5 mM PMSF, 10 mM Tris–HCL (pH 7.4)) and incubated on ice for 40 minutes. The lysates were then centrifuged (17,000 × g, 10 minutes, 4°C) and the post-nuclear fractions were transferred to new tubes. The protein concentration of each sample was determined by Bradford assay. Samples were adjusted with TBS buffer and 6× Laemmli buffer + 100 mM dithiothreitol (DTT) and heated at 50°C for 10 minutes. Samples were separated by SDS-PAGE and transferred to PVDF membranes (Merck), then blocked in 5% milk + PBS-T (PBS + 0.2% (v/v) Tween-20) for 1 hour. Blocked membranes were incubated with primary antibody (LRRC15 [LSBio aa393-422], ACE2 [Abcam Ab108252], GAPDH [GeneTex GTX627408]) in 5% milk + PBST (PBS + 0.2% (v/v) Tween-20) at 4°C overnight, then incubated with peroxidase (HRP)-conjugated secondary antibodies (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) [Jackson ImmunoResearch 111-035-144], Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) [Jackson ImmunoResearch 115-035-146]) for 90 minutes at room temperature.
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