I understand that the stainability of a protein with coomassie depends on the amino acid composition of the protein - how pronounced is this effect and is there a way to somehow introduce a correction factor in densitometry based on the known sequence and the amount of basic amino acids? (It seems that the arginine and lysine content is especially relevant).
Could anyone point me to some papers concerning this issue or could share experience?
Thanks in advance!
If you are trying to quantify a specific protein by densitometry readings from SDS-PAGE gels stained with Coomassie blue You can run a standard curve with the protein in question for comparative results against your unknown. The standard needs to be run with each unknown. The accuracy of the measurement will be +/- 10%. There is no mathematical calculation that I know of that correlates Coomassie blue staining intensity with protein concentration / composition. You need standards and should validate results determining standard error within and between assays.
Hello Peter, you may wish to have a look at the below paper!
Tal, M.; Silberstein, A.; Nusser, E. Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer. J. Biol. Chem. 1985, 260, 9976−9980.
Indeed you are right. The positively charged groups help in staining by electrostatic interactions, and modification of these residues e.g. by glycation of these residues may interfere with staining. Similarly, hydrolysis of proteins may result in peptides with different levels/or no charged residues in sequence limiting the use of this dye for any quantification.
Regards,
Anant Dave
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