We were doing western with cell lysate and used 5% BSA in TBST for blocking. Also, primary and secondary dilution of antibodies has been prepared in BSA in TBST. With this, we are getting a prominent non-specific band at 42 kDa while developing with any primary antibody.
The attached picture is a blot of PTEN (50-55 kDa), but the band is at 42 kDa. How can I get rid of this non-specific band?
Any kind of help will be appreciated.
Hi Liku Biswal
For difference in gel percentage, and in case of gradient gels, difference in gradient, and even difference in buffer systems can make your protein look wrong.
I would suggest you to check again for your buffer system and your gel what size the marker denotes.
Such clean bands cannot be regarded as non-specific.
Hope it helps,
Subham.
Did you really mean to say that you see the same band using any primary antibody? If that is the case then it sounds like your secondary antibody is reacting to something in your samples. You might want to try and see if that is the situation and if so then maybe you need to try a different secondary.
This is a problem with contamination in your secondary antibody, being either the secondary antibody itself is contaminated or the working aliquot is contaminated with another primary antibody (e.g. Actin). I would test this by making a new secondary antibody working aliquot and see if this bend persists. If so try a different batch of the secondary antibody.
Michael J. Benedik Thanks. I am getting the same band with multiple primary antibodies, even with those having molecular weights close to 42 kDa. Fresh secondary antibody was the first troubleshooting I did but faced the same thing. I will try using a secondary dilution from a fresh antibody vial.
Subham Basak thanks Subham. We are using the tris-glycine buffer and respective markers. and the attached image is of a 10% gel.
Is your protein prone to disulfide bonds? Does the loading buffer contains DTT? The bands look so good that I doubt it is coming from blotting. In my opinion or is either a contamination of your samples or maybe you need to add DTT to your sample or your protein is getting degraded somehow if it's a larger MW. We recently had an issue with some bands we thought were unspecific and when we sent them for analysis and it was out protein getting degraded.
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