The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ELISA: Use at an assay dependent dilution.
WB: Use at an assay dependent dilution (PMID 18611857).
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
Creatine Kinase MB consists of a dimer of nonidentical chains. With MM being the major form in skeletal muscle and myocardium, MB existing in myocardium, and BB existing in many tissues, especially brain.
Creatine Kinase MB reversibly catalyses the transfer of phosphate between ATP and various phosphogens. The creatine kinase isoenzymes play a central role in energy transduction in tissues with large fluctuating energy demands such as skeletal muscle, heart, brain and spermatozoa.
Western blot - Creatine Kinase MB antibody (ab31832)Image from Hurd TR et al, J Biol Chem. 2008 Sep 5;283(36):24801-15. Epub 2008 Jul 8, Fig S1.
The glutathionylated ~ 45 kDa band is sarcomeric mitochondrial creatine kinase, not a complex I subunit. Mitochondria (20 mg protein/ml) were first incubated with [35S]GSH (40
µCi/ml) in STE for 30 minutes on ice, pelleted, and washed in 250 mM sucrose, 1
mM EGTA, 10 mM Tris-HCl, pH 7.4. The bovine and rat mitochondria were incubated ± 0.5
mM diamide for 5 minutes at 37ºC and then with 50 mM NEM for a further 5 minutes.
Mitochondria were pelleted and complex I was isolated by BN-PAGE and
non-reducing SDS-PAGE and electrotransferred onto nitrocellulose. [35GSH]-
containing protein bands were detected using a phosphor imager. The membrane was blocked in PBST (PBS, 0.05% (v/v) Tween 20 with 1% (w/v) skimmed milk powder) and incubated with ab31832 at 1.25 µg/ml, for 1–2 hours at room temperature. Blots were incubated with 1/5000–1/10,000 dilutions of the appropriate secondary anti-serum for 1 hour at room temperature, treated with the chemiluminescent reagent (ECL or ECL Plus) and visualized on Super RX photographic film.
Hurd TR et al. Complex I within Oxidatively Stressed Bovine Heart Mitochondria Is Glutathionylated on Cys-531 and Cys-704 of the 75-kDa Subunit: POTENTIAL ROLE OF CYS RESIDUES IN DECREASING OXIDATIVE DAMAGE. J Biol Chem283:24801-15 (2008).
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