Cell lysates were diluted in Laemmli sample buffer (100 mM Tris–HCl, 2% SDS, 10% glycerol, 0.1% bromophenol blue) containing 5% β‐mercaptoethanol. After heating at 95°C, proteins were separated by SDS–PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat milk or 5% BSA and detection of individual proteins was carried out by blotting with specific primary antibody against MFN2 (ab56889, AbCam, Cambridge, MA, 1:1,000), CPT1 (GTX114337, GeneTex, 1:1,000), VDAC (Abcam, 1:1,000), Phospho‐DRP1 Ser637 (4867S, Cell Signaling, 1:1000), β‐actin (NB600‐501, Novus Biologicals, Littleton, CO 1:1,000), porin (ab61273, AbCam, Cambridge, MA, 1:1,000), and vinculin (V9131, Sigma‐Aldrich, 1:10,000). Proteins of interest were detected by chemiluminescence using a secondary peroxidase‐linked anti‐rabbit (1:10,000) or anti‐mouse (1:10,000) and a detection system.
Ab61273
Manufactured by Abcam
Ab61273 is a monoclonal antibody product manufactured by Abcam. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab61273
1
Mitochondrial Protein Expression Analysis
2
UCP3 Expression via Western Blotting
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The UCP3 expression was assessed by western blotting analysis, and the samples were normalised to VDAC protein. Mitochondrial protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1h, and incubated overnight at 4-8℃ with anti-UCP3 (1:1000, ab-3477,Abcam), and anti-VDAC (1:1000, ab-61273，Abcam) antibodies. [20] At fifteen days after birth,the tails of the mice were collectedand digested overnight at 55˚C by tissue cracking buffer containing protease K. DNA was extracted from the mice tailsas previously described. PCR were employed to genotype mice identification. The following primer sequences were used for detection: 5'CCT CCA CTC ATG ATC TAT AGA TC3', 5'GCA CTG CGG CCT GTT TTG3', and 5' ACC CTC TGT GCG CAC CAT AGT CA3'. The PCR products were analysed by agarose electrophoresis.
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