I did a western for the 65-67k Da proteins of interest. I have run western blot , but recently there were a lot of non-specific bands appearing. (There has been a one specific band several times before).
Hi there,
Do you follow exactly the same WB protocol with the same reagents?
A comparative picture should help to see where is the expected band on your WB...
As a first suggestion I would tell you to block the membrane in a better way to limit the non specific binding of the antibody and also to check the reactivity of the primary antibody.
HI,
There are numbers of reasons you could get multiple bands in your westerns. Given that you don't allow your samples to degrades while preparing leaves us with blocking and primary antibody specificity and concentration. As Dominique suggested try changing your blocking buffer. There quite a few options available( BSA, MILK, Fish serum or commercial ones). Also try diluting your primary and secondary antibodies little more. It also helps to dilute your Primary as well as secondary antibody in blocking buffer.
I also find keeping your transfer assembly real clean helps in a better transfer especially with wet transfer.
Hope this helps!
X-Ray film exposure time brings you a lot of variability in your results, as films only have a bandwidth of ~100 in intensity variation: Either you see nothing or too much.
What you can do is including a reference blot that is probed with the secondary antibody only, to ensure you can identify background signals.
Hi there,
There could be two main reasons for appearence of non-specific bands in your western. One is the improper blocking and second is the concentratoin of your primary antibody. Not sure, what blocking method and source of your primary antibody are. However, by the view of your western image, It appears that blocking could be defective, try using 5%BSA in TBS-T for one hour on a shaker/rocker. Secondly, as suggested, you may have to dilute your primary antibody bit more!
hi,
i am agree with the question raised by Kamil Krawczyński. are you using the same tissue or cell line or it is different. i am also concern about the sample preparation, are you using the same method or using a new method, because method of sample preparation is also very important ,and if it is a membrane protein then it is most important . another question i would like to ask is it the same lot of antibody you used previously?I hope these question may help you to solve the problem.
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