The cells were washed with phosphate-buffered saline (PBS) and lysed with cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, and Complete Protease Inhibitor Cocktail; Roche, Indianapolis, IN, USA) on ice for 30 min. Thirty to fifty micrograms of the cell lysate was resolved by SDS-PAGE on 10% or 12% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies followed by peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Calbiochem, EMD Chemicals Inc., San Diego, CA, USA) and the ECL reagent (Millipore) for band visualization. To verify equal loading and adequate transfer, the membranes were probed with anti-α -GAPDH antibodies (Santa Cruz Biotechnology). The primary antibodies were anti-STAT1, anti-phospho-STAT1, anti-STAT6, and anti-phospho-STAT6 (Cell Signaling Technology, Beverly, MA, USA).
Anti α gapdh antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-α-GAPDH antibodies are polyclonal antibodies that specifically recognize the α isoform of the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a widely expressed enzyme involved in glycolysis. These antibodies can be used to detect and quantify the α-GAPDH protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ecl reagent, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti stat6, by Cell Signaling Technology (1 mentions)
Anti phospho stat6, by Cell Signaling Technology (1 mentions)
Anti phospho stat1, by Cell Signaling Technology (1 mentions)
Anti ifnγ, by Santa Cruz Biotechnology (1 mentions)
2 protocols using anti α gapdh antibodies
1
Western Blot Analysis of STAT1 and STAT6
2
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Soluble lysates from the cultured cells were extracted in ice-cold SDS-lysis buffer [50 mM HEPES, 150 mM NaCl, 0.2 mM EDTA, 0.5% NP-40, 0.1% SDS, 1 mM Na3VO4, 10 mM NaF, and Complete Protein Inhibitor Cocktail (Roche)] for 30 min on ice. Thirty to fifty micrograms of the cell lysate was resolved by SDS-PAGE on 10% or 12% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies followed by peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (Ig) G secondary antibodies (Calbiochem, EMD Chemicals Inc., San Diego, CA, USA) and ECL reagent (Millipore, Billerica, MA, USA) for band visualization. To verify equal loading and adequate transfer, the membranes were probed with anti-α-GAPDH antibodies (Santa Cruz Biotechnology, Pasadena, CA, USA). The primary antibodies were anti-p50, anti-p65, anti-IFN-γ (Santa Cruz Biotechnology), anti-phospho-PKC isoform, anti-PKC isoforms (Cell signaling Technology).
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