Whole cell proteins were prepared by lysing cells in radioimmunoprecipitation assay buffer (Beyotime). Protein concentrations were determined by the BCA protein assay kit (Beyotime). Proteins (30 μg/sample) were fractionated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham, Piscataway, NJ, USA). Membranes were blocked with 5% skim milk for 1 hour at room temperature, then incubated with mouse anti-HLA-G (dilution 1:1500; Abcam Inc., Cambridge, UK) or mouse anti-GAPDH (dilution 1:5000; Abcam) primary antibodies overnight at 4°C. The next day membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody (dilution 1:5000; Abcam) at room temperature for 2 hours. Hybridized protein bands were detected with the enhanced chemiluminescence kit (Beyotime).
Horseradish peroxidase conjugated anti mouse secondary antibody
Manufactured by Abcam
Sourced in United Kingdom
Horseradish peroxidase-conjugated anti-mouse secondary antibody. A secondary antibody that binds to mouse primary antibodies and is conjugated to the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Foretinib, by Selleck Chemicals (1 mentions)
5 protocols using horseradish peroxidase conjugated anti mouse secondary antibody
1
Western Blot Analysis of HLA-G Expression
2
Western Blot Analysis of p53 Protein
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Briefly, the cells were washed with PBS and lysed. Protein products (8–10 µg/µl; 50–60 µg total) were separated using 10% SDS-PAGE and subsequently transferred overnight onto a polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked for 1 h with a Blotting-Grade Blocker (no. 1706404, Bio-Rad Laboratories, Inc.). The specific monoclonal p53 antibody (diluted 1:1,000; cat. no. ab1101; Abcam) was incubated overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:1,000; cat. no. 6120-05; SouthernBiotech, Birmingham, AL, USA). Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) was used to visualize the blots. The protein bands were exposed onto SuperRX X-ray film (Fujifilm Investment Co., Ltd.). Anti-GAPDH was used as a loading control (1:1,000; cat. no. ab9485; Abcam, Cambridge, UK).
3
Western Blotting of Extracellular Vesicles
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For Western blotting, EVs were lysed in 10× RIPA buffer (Millipore, Bedford, MA, USA) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Samples were identified under reducing (TSG101) and non-reducing (CD63) conditions, and 108 EV particles were loaded onto (10%) polyacrylamide gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane with a 0.45-μm pore size (Bio-Rad, Hercules, CA, USA) at 80 V for 2 h. After blocking with 5% skim milk, the membranes were incubated with anti-TSG101 (Abcam, ab83, Cambridge, UK) and anti-CD63 (MBL International Corporation, MEX002-3, Woburn, MA, USA) antibodies overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Abcam, ab6728, Cambridge, UK). ECL Blotting Reagent (Cytiva, MA, USA) was used for the chemiluminescence reaction. Images were analyzed using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).
4
Quantifying TNF-α Expression by Western Blot
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Cells were collected at 36 or 48 h after transfection, washed with PBS, and lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) followed by centrifugation at 5,000 × g for 20 min at 37°C for protein extraction. Proteins were quantified using a Bicinchoninic Acid kit, according to the manufacturer's protocol. Equal amounts of protein (20 µg/lane) were separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.), and blocked with 5% nonfat milk at 37°C for 1 h. After washing with TBS with Tween-20 (TBST), membranes were incubated with primary mouse anti-human TNF-α antibody (1:1,000; RayBiotech, Inc., Norcross, GA, USA; cat. no. 119–1633) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:2,500; Abcam, Cambridge, UK; cat. no. ab97046.) for 3 h at 37°C, and developed with enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). Expression levels of TNF-α were analyzed with β-actin (1:2,500; Abcam; cat. no. ab8227) serving as an internal control.
5
Foretinib Signaling Pathway Evaluation
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Foretinib (>99%) was purchased from Selleck (Houston, TX, USA). Dulbecco’s Modified Eagle Medium (DMEM), the media were purchased from Thermo Co., Ltd. (Waltham, MA, USA), and fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Primary antibodies against p-MET, HGF, glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-Actin and horseradish peroxidase-conjugated anti-mouse secondary antibodies were obtained from Abcam (Cambridge, UK).
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