Assay of PKM2 dimer and tetramer in vitro follows previous descriptions41 . In brief, after 40ng PKM2-FBP complex was treated with 200–300μM SNO-CoA for 10 min at room temperature, 5 μl fresh glutaraldehyde (50%) was added to a reaction mixture containing 100mM HEPES (pH 7.5) for 5 min at 37°C. The cross-linking reaction was terminated by addition of 5 μl 1M Tris-HCL (PH 8.0). Assay of PKM2 dimer and tetramer in situ was carried out as described previously14 (link). DSS (disuccinimidyl suberate; Thermo Scientific) (final 500 μM) was added to cells for 30 min at room temperature to cross-link proteins. Cells were lysed in RIPA Buffer and protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by 4–20% Criterion™ Precast Midi Protein Gel (BIO-RAD) and monomer, dimer and tetramer forms of PKM2 were detected with PKM2 antibody (sc-365684, Santa Cruz Biotechnology).
Pkm2 antibody
Manufactured by Santa Cruz Biotechnology
The PKM2 antibody is a laboratory tool used in biological research. It is designed to specifically detect and bind to the PKM2 protein, which is a key enzyme involved in cellular metabolism. This antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and distribution of PKM2 in different cell types and biological samples.
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2 protocols using pkm2 antibody
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In Vitro and In Situ Assay of PKM2 Oligomers
2
In Vitro and In Situ Assay of PKM2 Oligomers
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Assay of PKM2 dimer and tetramer in vitro follows previous descriptions41 . In brief, after 40ng PKM2-FBP complex was treated with 200–300μM SNO-CoA for 10 min at room temperature, 5 μl fresh glutaraldehyde (50%) was added to a reaction mixture containing 100mM HEPES (pH 7.5) for 5 min at 37°C. The cross-linking reaction was terminated by addition of 5 μl 1M Tris-HCL (PH 8.0). Assay of PKM2 dimer and tetramer in situ was carried out as described previously14 (link). DSS (disuccinimidyl suberate; Thermo Scientific) (final 500 μM) was added to cells for 30 min at room temperature to cross-link proteins. Cells were lysed in RIPA Buffer and protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by 4–20% Criterion™ Precast Midi Protein Gel (BIO-RAD) and monomer, dimer and tetramer forms of PKM2 were detected with PKM2 antibody (sc-365684, Santa Cruz Biotechnology).
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