Hi, I am using serum samples for western blotting. I have used different antibodies (for different target proteins) and most of them are monoclonal. I am getting unusual results like a band around 50kDa in almost all samples irrespective of the antibody.
As I am getting that band, I have doubts about my target protein also, a few of which are around 50+/-5 kDa.
How is that possible and how can I get rid of that?
Have you performed albumin depletion for the samples before SDS-PAGE? If not the band you are observing in all your samples could be albumin. It would appear as a blotched band around 66-50kD. You will not have proper separation of proteins if you do not deplete the serum of albumin and other prominent proteins like immunoglobulins. You can follow the TCA-Acetone method for the same. If you could post an image of your blot, I can confirm the same. Hope this helps.
Have you performed albumin depletion for the samples before SDS-PAGE? If not the band you are observing in all your samples could be albumin. It would appear as a blotched band around 66-50kD. You will not have proper separation of proteins if you do not deplete the serum of albumin and other prominent proteins like immunoglobulins. You can follow the TCA-Acetone method for the same. If you could post an image of your blot, I can confirm the same. Hope this helps.
Hi Sandeep.
If the serum has been prepared correctly, the platelets should remain in the plasma I think?
I agree with Akhil, it could be albumin.
In addition, another common contaminant is keratin, which is around 60 kDa. This can be avoided by being extra careful when handling/running the gels, etc. (A quick google can provide you with more tips :) )
@akhil i didn’t deplete albumin.
Will try that...appreciate if you can provide an validated link for the same.
Article Optimisation of a serum albumin removal protocol for use in ...
@ Sandeep Rai Kaushik Please try this method. You might have to standardize the ratio of sample to TCA/Acetone reagent. Though the protocol is for equine samples we are using this protocol successfully in our lab for human serum samples.
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