Proteins were extracted from cells using RIPA lysis buffer (Solarbio, Beijing, China) containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA protein quantification kit (Solarbio, Beijing, China). Equal amounts of protein (20 µg) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (#ISEQ00010, Millipore, Billerica, MA, USA). Membranes were then blocked in blocking buffer (TBST solution with 5% skim milk) for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following day, the membranes were washed. Then, the membranes were incubated with the secondary antibody (1:5000) for 1 h at room temperature. Protein bands were visualized using an ECL kit (#P0018M-2, Beyotime, Beijing, China), with β-actin serving as the internal loading control. Protein expression was analyzed using Image J software (version 2.3.0). The primary antibodies used included anti-β-actin (Proteintech, Chicago, IL, USA), anti-SRSF7 (Proteintech, Chicago, IL, USA), anti-PKM1 (Proteintech, Chicago, IL, USA), anti-PKM2 (PTM BIO, Hangzhou, China), anti-LDHA (Proteintech, Chicago, IL, USA), and anti-GLUT1 (Proteintech, Chicago, IL, USA).
Anti pkm2
Manufactured by PTM Biolabs
Sourced in United States
Anti-PKM2 is a laboratory product used to detect and quantify the expression of the PKM2 protein. PKM2 is an enzyme involved in the glycolytic metabolism of cells. Anti-PKM2 allows for the analysis of PKM2 levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Iseq00010, by Merck Group (2 mentions)
Anti ldha, by Proteintech (1 mentions)
Anti pkm1, by Proteintech (1 mentions)
Anti β actin, by Proteintech (1 mentions)
β actin, by Beyotime (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Bca protein quantification kit, by Solarbio (1 mentions)
Anti glut1, by Proteintech (1 mentions)
P0033, by Beyotime (1 mentions)
Sds page sample buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti β actin, by ZSGB-BIO (1 mentions)
2 protocols using anti pkm2
1
Immunoblotting Analysis of Metabolic Proteins
2
Protein Extraction and Western Blotting Protocol
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Total proteins were extracted according to the manufacturer’s instructions (P0033, Beyotime). The total protein concentration was determined using a BCA kit (P0010, Beyotime). The proteins were then dissolved in 5 × SDS-PAGE sample buffer (P0015, Beyotime) and heated to 99 °C for 10 min. Equal amounts of proteins were loaded into each well of polyacrylamide gels (6%–15%, Epizyme Biotechnology) for electrophoresis, and then transblotted onto a 0.2-μm polyvinylidene fluoride (PVDF) membrane (ISEQ00010, MerckMillipore). After being washed three times, the membranes were blocked with QuickBlockTM blocking buffer for Western blotting (P0252, Beyotime) at room temperature for 30 min. The blots were then incubated with anti-LETM1 (1:4000, Proteintech), anti-G6PD (1:1000, PTM BIO), anti-PKM2 (1:2000, PTM BIO), anti-OPN (1:2000, ZEN BIO), anti-Osteocalcin (1:500, Biorbyt) or anti-β-actin (1:4000, ZSGB BIO) antibodies at 4 °C overnight, and then with horseradish peroxidase-linked anti-mouse or anti-rabbit secondary antibodies (1:10,000, ZEN BIO) at room temperature for 2 h. The bands were detected with a chemiluminescent horseradish peroxidase substrate (NCM Biotech). The expression of β-actin was used as a loading control.
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