Hi all,
Is it possible that the migration of my protein is delayed due to the complexity of the sample?
When I run a western blot with 1) a protein extract from locust tissue and 2) hemolymph (can be considered as the insect's blood) sample, I detect my protein in both samples but the band detected in the hemolymph is a bit higher located than the one I detect in the protein extract.
My hypothesis is that the migration in the hemolymph sample is a bit slower due to its complex nature?
Many thanks!
Separation of proteins in SDS page method is according to MW therefore you think wrong.
There is difference between your pro.
Regards.
Do all the other proteins run normal? Specifically, the impurity bands in your sample lane, do they run normal or can they also be found at a slightly higher molecular weight?
What approx molecular weight have your protein? And what percentage of acrylamide are you using in your gels?
It is not a problem of complexity. Perhaps it could be a problem of protein aggregation. It is possible you are loading too much protein in your samples. Try again diluting the sample.
Hi all,
Thanks for your answers.
@ Achim: I haven't checked this yet, I was thinking to repeat the blot and read out another protein (if there is again a difference in MW between tissue and hemolymph), then I'm sure there is a different migration.
@Maria Luisa: It's around 96kDa. In hemolymph, I detect a band a bit higher, in tissue, I detect a band a bit lower. Percentage is nupage bis tris 4-12%.
Take in mind, most proteins secreted by cells before secretion have a signal peptides in cell to secrete right therefore is usual you have higher MW of this type pro in tissues.
I think its not, due to the basic principle of SDS which separate the protein based on its molecule weight, so how complex your sample, it will finally separate diferently in different distance because they have a different molecular weight. Maybe for make sure, you can run your sample with another sample with a different complexity so you can make sure your hypothesis :)
Oh and now Im doing my research using SDS Page too, so hopefully we can share our experience in our own research thankyou :)
Run both samples separately on the same gel and also a third lane which has the control in which both samples are mixed together . if you see 2 bands in the lane containing a mixture of both samples on Western Blot then the proteins have different MWt.
There are some possibilities. The protein in the hemolymph may have different post-translational modification, giving it a higher MW. Or, the protein in the hemolymph may be an isoform (with higher MW) compared to the main isoform in the whole tissue sample. Another possibility is that, when you prepared the whole locust tissue extract, the protein of interest was exposed to a proteolytic enzyme that digested it partially and thus reducing the MW, and that proteolytic enzyme was not in the hemolymph.
Anna Tan-Wilson's comment concerning proteolytic digestion is a good point.
Try adding some protease inhibitor mix; at a mininimum, try adding EDTA, as it will inactivate the abundant metallo-proteinases.
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