two bands on WB-alternative use of start codons?
Posted 20 August 2012 - 04:48 AM
I have a question regarding detection of one protein using Western blot. Antibody we use gives us two bands when only endogenous proteinis present and the same two bands when there is exogenous protein present in cells (after transfection). Since there are two start codons in the gene which encodes this protein, we were wondering is there any way to determine if these two bands are from alternative use of start codons? Any other way except for Edman sequencing?
Posted 20 August 2012 - 09:58 AM
How far apart are these two bands? Maybe you have a post-translational modification: phosphorylation, ubiquitination etc. which is more probable. or 2 splicing variants? I would go for Edman sequencing or MS. But both of them wouldn't get you too far. Edman sequencing works only for the first few amino acids in the sequence and MS gives you some peptides that you have in your protein. Both of them would not tell you too much about the full sequence or post-translational modifications.
Posted 20 August 2012 - 01:48 PM
If you have cloned sequence, you could delete/mutate one of the start codons and see if you still get 2 bands.