I am doing western blotting for a protein(mol weight 77kD). It is glycosylated at 4 amino acids. Company spec sheet shows a band at 100kD. They have done it in rat brain homogenate. But I have tried in mouse retinal samples and getting a single band near 75kD. How do I confirm that the band which I am getting is correct? Is it possible that the band which I am getting is of non glycosylated protein?
Assuming that the protein is not always glycosylated, that's a possibility (plus, there may be different glycosylation patterns between mice and rats, no?). If you have access to a glycosylation cleaving enzyme you could try treating your sample with that to see if the MW changes.
Assuming that the protein is not always glycosylated, that's a possibility (plus, there may be different glycosylation patterns between mice and rats, no?). If you have access to a glycosylation cleaving enzyme you could try treating your sample with that to see if the MW changes.
Thanks Krzysztof and Lauren..But I am wondering if a single protein can be of different molecular weight in two different tissues. Actually I doing western for synaptic vesicle protein (MW 77KDa). According to manufacturers detail, one should get a band at 100kDa because of glycosylation at 4 different amino acids. They have used rat brain homogenate. Same antibody when I am using for mouse retina, I am getting a band below 75KDa. I am looking for a possible explaination.
I am by no means an expert on post translational modifications (PTMs), but I think that its possible to have different PTM events (including an absence of them) in different tissues/under different conditions. You may also have different PTM events between the two species, no? Try taking a look at this page: http://www.piercenet.com/browse.cfm?fldID=7CE3FCF5-0DA0-4378-A513-2E35E5E3B49B
Another thought: have you checked the sequence homology? Perhaps the mouse protein is smaller or lacks one of the glycosylation sites? Given the difference in species and tissue types, there could be different post translational modifications....
Given that your protein is running at a lower MW than expected (
Thanks a lot Lauren..I do use protease inhibitor.
I had written to the company.They repeated the experiment with retinal samples and got results similar to mine. So I guess half of the problem is solved. But why we are getting this band at lower position is still not cleared. They say that It might be because of differential glycosylation...
I'd agree with the company and speculate that the different glycosylations between species could play a role.
When you say that your protein is
Hey Lauren, Thanks. I am getting protein band at slightly lower than 75KDa. The observed size is in mice and 77kDa is predicted from the sequence. I too feel that its all because of differential glycosylation in different tissues. I will confirm this by using glycosylation cleaving enzyme. Thanks, your suggestions really helped me a lot..
If you have produced in vivo your protein, the glycosilation level is variable and you will have a mix of different glycosilated species of your target protein, therefore you will see something like a smear in your SDS-PAGE and some of the glycosilated species will have a Mw much higher of the theoretical Mw of the a-glycosilated specie, it will depend on the glycosilation grade obtained.
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