Western blotting - (Jan/22/2013 )

Hi every one,

I am doing my research on Helicobacter pylori. After lyse the infected mammalian cell, I am doing western blotting to check the injected virulent protein. for that I am using rabbit polyclonal abs, and am experiencing non specific binding and lot of noise background, mean while i didnt experience any other non specific binding while using monoclonal abs for other targets.

Am doing 1:30 hrs membrane blocking, abs incubation for over night, 1:30 hrs abs blocking and 1:30 hrs secondary ab incubation with 3x10 min washing. some times i didnt got any noise but many times i am getting noise background and non specific binding.

How to overcome this problem.

Hope for looking valuable suggestions.

thanks

micronagu

micronagu on Tue Jan 22 13:49:51 2013 said:

your primary and secondary antibody dilutions? You can also reduce the incubation of the secondary to 45 min to an hour. For a comparison between polyclonal and monoclonal antibodies, check this out: http://www.abcam.com/index.html?pageconfig=resource&rid=11269&pid=11287

I would also add - what is in your antibody incubation buffer? If you add 0.1-0.5% tween 20 and some BSA or other protein you will likely improve the specificity of the binding.

bob1 on Tue Jan 22 20:20:38 2013 said:

Could you explain how tween and BSA will improve specificity? I've always wanted to know the reason since i use tween 20 but not BSA.

thanks to every one for your valuable reply.

actually my primary ab - 1:2000, secondary - 1:3000, and I am using 10% skim milk in 0.05% Tween 20 in PBS to dilute the abs.

science noob on Tue Jan 22 23:42:56 2013 said:

Tween partially denatures the antibody by separating the chains (IIRC). BSA acts as a non-specific blocker which blocks non-specific sites.

micronagu on Wed Jan 23 05:08:08 2013 said:

You can cut the skim milk down quite a lot 1-5% is fine. If you raise the % tween, to 0.1% it might improve too. IF you are detecting a phospho-protein then don't use milk as the block, as it is absolutely full of phospho proteins.

bob1 on Wed Jan 23 08:19:45 2013 said:

Does this principle only apply to WB or would this also be worth a try in other antibody-dependent methods ? (never heard of this for Flow Cytometry, for instance)

also, no need to block again after incubating with the primary antibody (you may be leaving some primary antibody behind, this will increase background). just perform washes as you do after the secondary antibody.

Tabaluga on Wed Jan 23 09:03:24 2013 said:

Works for any antibody based method where you need specific binding... However, if you have a protocol that works and you are happy with it, I wouldn't recommend changing the conditions. Usually I have found that titration of the antibody and conditions to give a specific band on a WB is the most important step in this process, and that the conditions used in a WB should be transferrable to IHC/IF but aren't necessarily.

thank you very much for your kind reply, and i will work on it..