non-specific binding in western blotting - non-specific binding in western blotting (Sep/03/2007 )

Hi Everyone,

I am getting problems with non-specific binding in western blotting. After developing I can see almost the same type of bands as I see after ponceau staining. This time I doubled my antibody concentration, as using the specified concentration gave no band.

Please somebody help me out.

Thanks and Regards
SR

-sweta rani-

How do you block? Please give your conditions, so we can look if you do it too short or with too less blocking agent.

-biomaus-

QUOTE (sweta rani @ Sep 3 2007, 10:45 AM)

What kind of antibody do you use?
monoclonal or polyclonal?
self clean up or bought?
from which company?

-kylvalda-

QUOTE (sweta rani @ Sep 3 2007, 01:45 AM)

too high titer of primary or secondary or both Ab´s; use secondary with 1:1000; optimize then conc of primary Ab

-The Bearer-

Do you have a positive control loaded?
- It is easier to optimise if you have a positive control.

Are all the bands of the same intensity?
- I find it more difficult to gain a clean signal when there doesnt seem to be a clear band of your desired sized among other non specific bands.

How long are your incubations for blocking, primary etc?

-Tyro-

what is your washing solution? do you include tween?

-mdfenko-

try different blocking solutions to decrease unspecific binding.

Also keep in ming, the antibody might not be specific.

-scolix-

Hi Everyone
I tried to alter time of blocking and now I am getting band with the positive control but not for my sample. I tried using different lysis buffer but it dosent work. However I did immunofluorescence and it works there, but no luck with western.

Could it be possible that there is very samll amount of protein and is beyond the detection limit of western. I am able to get signals from qRT-PCR.

Regards
sweta

-sweta rani-