I separated proteins from porcine muscle tissue, and I noticed that three spots from CK-m type protein were resolved in around 39 kDa. My question is there any species or modified form of this protein that is lower than 43 kDa?
Probably non-glycosylated...That also depends of your separation (2D or Mw) approach, i.e. gradient gel or some % of acrylamide. CK-m shows to appear at lower size in 12% AA handcast ge, but this is probably due to low resolution power of separation in that Mw range. If it was good separation of your protein spots then stain your gel for glycoprotein detection. Second thing is what MS data gave you? Check the blank which was run between two samples, maybe is contamination from previous sample.
Good luck!
Dear Eric,
it depends on your sample, from rabbit it should be 43kDa (http://www.uniprot.org/uniprot/P00563), if the size is different there could be some postranslational modification, but I think that you should see that one with and without modification.
Next, Creatine kinase M-type acts in some tissues as homodimer or heterodimer.
Regarding the MS data, be aware of right interpretation if you use LC-MS, because this analysis can show trace amounts in other spots.
FINALLY - be sure that the theoretical size from MS search is not precursor, but the expected size of the protein !
some time ago, I have analyzed these proteins in 2D-E (sample from Oryctogalus cuniculus muscle), see Figure 5 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022860
Success ;-)
The quantity of SDS bound by proteins is usually about 1.4g of SDS /g polypeptide. However it is possible that this quantity varies from protein to protein, depending on their amino acid sequences, and this could affect their mobility in SDS-PAGE. In addition some proteins have a net charge which is significantly different from the average charge on proteins. Some are very basic and some very acidic. Potentially this could affect the overall charge to mass ratio on the SDS/ protein complexes and this in turn could affect their electrophoretic mobility. One example of a protein that exhibits anomalous SDS-PAGE mobility is rabbit muscle Troponin T which has a reported MWt of 37kDa as measured by SDS-PAGE compared with a 30,500Da as determined from its amino acid sequence .
References:
Wilkinson, JM., and Grand, RJ., (1975) Proc. FEBS Meet.9th 31,137-144.
Pearlstone JM, et al., J Biol Chem. 1977 Feb 10;252(3):983-9
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