Hello Nand, I presume you are doing Western blots with a crude cell lysate. A few questions for you, is there a possibility of purification before the blot? If not, it would have been better to decrease you antibodies concentrations.

A suggestion: can you make available the protocol you used.

Regards

Thank you Robert for the reply.

Yeah, I am using the crude cell lysate. At present I don't have an option of purification.OK i will decrease the concentration, but I am afraid if i will loss my protein of interest also. 

I am attaching the protocol which I followed.

What is the size of your protein?

I would use a dounce homogenizer to get better extraction of proteins from the cells.

Do not boil the samples, heat to a max of 90oC, but I would also try not heating at all since heating can activate proteases even is SDS which can unfold proteins and make them more susceptible to cleavage!

Thanks Gary, My protein size is around 60kDa. Before loading the protein I use to heat  at 95oC for 5 minutes for denaturation.

Sometimes the nonspecific bands are produced by the secondary antibody binding to very abundant proteins.  You could blot with secondary alone to test for this, or use more dilute secondary antibody to see if your blot looks any better (I dilute HRP secondary 1:10,000, but this depends on the supplier).  You might also try blocking with BSA instead of milk.  However, the problem may simply be that the antibody you are using recognizes a lot of proteins in addition to the one you want to detect (especially likely for polyclonals).  Unless the affinity for the undesired interactions is substantially worse than the desired ones, changing the blotting conditions won't fix this.   If you can't purify the protein, you might affinity purifying the antibody.  Otherwise see if there is another antibody available.  For commercial antibody, it's unfortunately common that they aren't as specific as shown in the catalogs.

Thanks Michael for sharing your insights.

You can try to do immunoprecipitaion for the protein of interest with the antibody and then blot with the same antibody, that helps concentrate the protein of interest and you might get rid of unspecific bands. Add an IgG as negative control and try Ripa buffer to start with, it's harsh but get's rid of background. 

Hi

The problem is the presence of non-specific IgG in your antibodies solution, reacting with corresponding antigens. You can remove them by antiserum absorption and subsequent IgG isolation. You can also perform a preliminary optimization test using different dilutions of antigen, primary and secondary antibodies with different times of treatment on nitrocellulose membrane as a dot immunobinding test, determine the best combination and then perform your western blot test.

Regards

Thanks Rasoul

Hi, I would suggest to increase your blocking time with BSA or milk before you add the antibodies. As already suggested the second thing could be to play with the dilutions of primary as well as secondary antibodies.

Good luck

Thanks Rakesh for the response.

Hi Nand,

In our lab, after the protein is transferred to the membrane, we wash membrane with PBST 3x10 minutes before blocking with primary antibody. Then we would wash membrane in 4% milk (made in PBST) 3X10 minutes before adding secondary antibody. After that, we will wash the membrane in PBST 3X10 minutes before exposure. Hope this can help you get a better image.

Good luck.

Thanks Guo for the response.

By the way, do you have a positive control for your protein of interest? Just wanna make sure the antibody is working well.

Sadly, I don't have any positive Control Guo.