dear Sara Kabir

Since Western blotting detects even very small amounts of protein, even a small amount of leakage from the side wells can cause false positives and errors. Therefore, I suggest that you place an empty well between the test wells and try not to fill the wells completely. In addition, be careful not to spill even a little protein on the top of the gel buffer. (in this case you will get a narrow false positive band through all your wells)

I can help you better if you attach a photo of your gel. Because by observing the type of band in the SDS PAGE and the result of the western blot, it is possible to recognize the type of error to some extent.

By the way, of course you should check your host to see if it carrying the GST gene. Because the antibody does not recognize whether the GST it is recognizing is a host product or produced by you ;)

thanks Masoud Kh! I think we got the idea. we tried the empty well technique in between and it worked. so I guess there was some leakage going on since we are using lab made polyacrylamide gels.

dear Sara Kabir

I am glad that your problem is solved.

Good luck