Sorry - can you please explain what you mean by indigenous antibody? Make sure you are blocking your blot appropriately - at least 1 hr at room temp with 5% non-fat milk (or use BSA). Are you sure the other bands picked up by your antibody are non-specific? Could they be smaller isoforms of your target protein, but with the same epitope? Could they be degradation products? Those are some things I would consider.

Sorry - can you please explain what you mean by indigenous antibody? Make sure you are blocking your blot appropriately - at least 1 hr at room temp with 5% non-fat milk (or use BSA). Are you sure the other bands picked up by your antibody are non-specific? Could they be smaller isoforms of your target protein, but with the same epitope? Could they be degradation products? Those are some things I would consider.

Is your antibody a monoclonal or polyclonal? A polyclonal antibody may not be pure. In other words, it may be a mixture of antibodies recognizing different epitopes. Can you affinity purify your antiserum with the target of interest?

I agree with the above suggestions.

moreover,you may add a negative control cell lysate, in that case, if these bands were also positive, they were non-specific binding, if not, they were degraded products of your target peotein.

Hi there,

Increase the stringency during incubation with antibody and during washes.

The antibody was raised in the lab and purified. Its poly clonal. Increasing the stringency did not eliminate the bands. May be next time , I can add a negative control. Thank you for your suggestions

You could also try to do a preadsortion of the antibody with a negative control protein extract (the one you would use in your western). 

This probably will reduce your non-specific bands

I see sometimes the same problem (non-specific binding) depending on the antibody. Even though you raise the polyclonal antibody by purified target protein of interest, many unrecognized impurity in the antigenic protein mixture generate antibody which cause non specificity. But you can see the results if the non-specific bands are absent around the relevant position in the control samples. As an another Dr. recommend, it is better rely on monoclonal antibody or affinity-purified polyclonal antibodies.

Hi Jaya, in cases where the target is a glycoprotein, glyco-epitopes can share significant structural homologies beyond the limits of protein families and they are responsible for extensive cross-reactivity. Such cases are particularly common when working on plant glycoproteins which carry carbohydrate determinants that do not exist in mammals, thereby exhibiting high immunogenic potency and giving rise to antibodies such as IgG and IgE. To prevent this type of interactions, you can try to inhibit non-peptidic binding sites by the pre-incubation of your diluted sera/antibodies with a mixture of 10 mM α-D-methyl mannopyranoside and 100 mM α-D-methyl glucopyranoside during 30 min to 1 h and see whatever it happens…

Add blockers, such as BSA, milk, fetal calf serum, the serum from the animal species, used to generate the antibodies, MgCl2, detergents such as the Tween or Triton-X family, block polymer detergents.

If possible, block the antibody with cell extracts of negative control in addition to BSA/skim milk.

Thanks for the suggestions.

I have two suggestion for

1. You can reduce the Incubation time primary antibody.

2. You can increase the detergent in your antibody binding buffer.

in truth and in my experience Western blots are not as easy to optimise in terms of stringency and specificity as Northern and Souhern blots. The single most effective thing you can do with a western blot when non specific bands are present finances permitting is change the antibody/source of antibody. I had one particular Western blot where I was obliged to try 3 different antibodies before obtaining a blot with a single specific band in spite of published claims. This is one reason I suspect that companies will allow you to trade one ab in for another free of charge if the original purchased ab does not work as claimed

The problem with mine is that its produced in the collaborators lab, not commercially available which is polyclonal which further increases the cross reactivity

Maybe you can try antigen-purification of your antibodies? The simple protocol by Ed Rybicki, which I personnaly found very easy and producing good results you can find here:

http://www.mcb.uct.ac.za/Manual/monospec_antibodies.htm

If you have pure antigen you may use also dot blot or just soak NC in antigen before purifying antibodies.

Good luck!

thank you. let me try it out