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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


first Antibody

first Antibody - Western Blot Forum

first Antibody - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 01-08-2013, 08:02 PM
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Default 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?
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Old 01-18-2013, 04:01 PM
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Default Re: first Antibody

bump up :-(
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Old 01-19-2013, 05:51 AM
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Default 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.
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Old 01-19-2013, 08:16 PM
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Default 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?
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Old 01-20-2013, 03:43 AM
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Default 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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