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-   -   first Antibody (http://www.molecularstation.com/forum/western-blot-forum/87286-first-antibody.html)

DannyB. 01-08-2013 08:02 PM

first Antibody
 
hey, how does the first antibody recognize a specific epitope of my Protein on the Membrane after it is linearized because of the SDS in the Gel? :newbie:

DannyB. 01-18-2013 04:01 PM

Re: first Antibody
 
bump up :-(

butters 01-19-2013 05:51 AM

Re: first Antibody
 
SDS-PAGE (I assume this is what you did) transfer to membrane, then proceed to western blot. If you use this method generally your protein may linearized, first antibody generally is your sample serum (antibody produce when reacted in vivo with whole or lyse bacteria / antigenic site).

Some antigenic protein doesn't work on denatured (SDS) gel but work in native gel. You can read up on Laemli's PAGE for more detail.

DannyB. 01-19-2013 08:16 PM

Re: first Antibody
 
thx for an answer.

so this means the Protein on the Membran (after SDS-Page and transfer) is still linearized? And the first antibody does not bind to the native Protein, instead it binds a specific sequenze of my Protein?

butters 01-20-2013 03:43 AM

Re: first Antibody
 
If you use SDS in your gel most likely it is linearized. Do take note that antigenicity is not protein alone not on all case, it could be glycoprotein (sugar+protein).

If you check back at your transfer buffer, does it have SDS?

My suggestion would be to talk with your boss or workmate as I am clueless what protein are you looking at.

Have you read up on Laaemli yet?


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