Antibody-Based Technologies => Antibodies Review => Topic started by: junfengwang on January 08, 2010, 12:17:56 PM

Title: Antibody for western blot and immunohistochemistry
Post by: junfengwang on January 08, 2010, 12:17:56 PM
We have one antibody that produces a beautiful single Western blot band. However when we use the same antibody to perform immunohistochemistry, we can not get staining even we use relatively high concentration of antibody.   I do not know why it is in this case.   
Title: Re: Antibody for western blot and immunohistochemistry
Post by: Cardio on January 08, 2010, 12:39:46 PM
Whenever you buy antibodies the datasheet will tell you what the antibody has been tested for. Some ab's work well with westerns and not at all on sections. Its always best to check this on the companies website before you purchase the ab's.

Title: Re: Antibody for western blot and immunohistochemistry
Post by: junfengwang on January 08, 2010, 06:41:36 PM
Thank you for your reply. I just wonder what is the mechanism behind this.
Title: Re: Antibody for western blot and immunohistochemistry
Post by: Cardio on January 09, 2010, 01:43:56 PM
There could be a number of reasons.
In a western you have denatured protein and no fix. The protein is also concentrated into a thin band. By denaturing the protein the epitopes are generally available to the ab and without the fix the masking of the protein is normally not a problem.
On sections though you have to one make the protein available through detergents and find out what fix is compatible with the ab's. Some fixes cross link and cover up the epitope and others can change the confirmation of the protein also masking the epitope. Given that each protein has a unique make up you can easily see how this can get complicated.
These are just few explanations but its best to do your homework on each antibody before you purchase it.
Title: Re: Antibody for western blot and immunohistochemistry
Post by: junfengwang on January 13, 2010, 04:39:24 PM
Thank you very much. It is very helpful.