I overexpressed soluble cytochrome c in e.coli. After Ni-NTA. I am getting two protein bands on SDS-PAGE - the one which matches my theoretical protein molecular weight 40 kda is having a fainter band(upper band) than the other band which is 33kda(lower band)? With the mass spec analysis , both bands contains the similar number of peptides from the same protein.
What could be the possible reasons?
Thankyou
Well, youe protein could be undergoing proteolysis thus resulting into a lower molecular weight band of 33 kDa.
Run sample after sonication or lysis of the cells to find out whether cleavage occurs in the cell or after lysis. Centrifuge sonicated sample at high speed ( i. e 17 000g) and run the supernatant for soluble proteins.
Always a good idea to add a protease inhibitor (EDTA-free) or PMSF. And also purify protein as quickly as possible. Some proteins require to be purified cold.
You can do N-terminal sequencing on both protein bands from the SDS-PAGE gel to find out which peptide bond is being cleaved.
Also, as the difference between the upper and lower band is 7 kDa, you can run a 20 - 22% SDS-PAGE gel to see whether you'll find the cleaved fragment - after sonication or purification (Ni column).
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