two bands on WB-alternative use of start codons? - (Aug/20/2012 )

Hello!

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?

Thanks!

This is unlikely because you would also need a proper Shine-Dalgarno sequence in the proximity of the second ATG to get this activated as a start codon.... which nature does not allow (in the assumption you are using a natural sequence not a synthetic one) If any ATG codon would be recognized as a start codon that would mean that the proteins would not be allowed to have methionines. There are programs to check for Shine-Dalgarno sequences in a gene. Maybe first you try this.

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.

Andreea

To add to Andreea's comments: It may well also be that the antibody has a non-specific band (actually this is very likely). If you have peptide for or know the epitope of the antibody you can do peptide competition to determine if one of the bands is non-specific.

If you have cloned sequence, you could delete/mutate one of the start codons and see if you still get 2 bands.