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native Vs SDS PAGE - (Feb/07/2008 )

hi,i am new in this forum and in the field of research as well.i hope anybody will help me out ? actually i have to check the antigenicity of some proteins from my sample.so should i go for native PAGE or SDS PAGE. will boiling during sample preparation affect the antigenicityof proteins?

thanks in advance! rolleyes.gif

-srianki-

What do you mean that you want to check the antigenicity of the protein?


SDS PAGE will allow you to test if the antibody is present in the sample. Boiling would denature the protein but preserve the protein for detection.

-scolix-

you need to know if the antibody will work with native and/or denatured protein. this depends on the epitope to which it is specific. if the antibody is polyclonal then it should work with both.

then you have to decide what you want to show and which gel type will show it best.

-mdfenko-

QUOTE (mdfenko @ Feb 7 2008, 11:33 AM)
you need to know if the antibody will work with native and/or denatured protein. this depends on the epitope to which it is specific. if the antibody is polyclonal then it should work with both.


I find this statement interesting for educational purposes. Are you saying that polyclonal antibodies should always (or almost always) work for both native and denatured proteins? What about the case where the antibody was raised against a denatured protein -in other words, the antigen was isolated from an SDS gel and then injected into the rabbit? Would you then still expect it to detect a native protein?

Thanks for the insights.

-smu2-

QUOTE (smu2 @ Feb 7 2008, 02:03 PM)
QUOTE (mdfenko @ Feb 7 2008, 11:33 AM)
you need to know if the antibody will work with native and/or denatured protein. this depends on the epitope to which it is specific. if the antibody is polyclonal then it should work with both.


I find this statement interesting for educational purposes. Are you saying that polyclonal antibodies should always (or almost always) work for both native and denatured proteins? What about the case where the antibody was raised against a denatured protein -in other words, the antigen was isolated from an SDS gel and then injected into the rabbit? Would you then still expect it to detect a native protein?

Thanks for the insights.

Thanks for replying .actually i have some proteins which i would separates on gel then inject to animal after cutting the respective bands.but i don't have to raise the antibody.so i want to know whether denaturing will affect my protein's antigenicity.

-srianki-

QUOTE (smu2 @ Feb 7 2008, 05:03 PM)
I find this statement interesting for educational purposes. Are you saying that polyclonal antibodies should always (or almost always) work for both native and denatured proteins? What about the case where the antibody was raised against a denatured protein -in other words, the antigen was isolated from an SDS gel and then injected into the rabbit? Would you then still expect it to detect a native protein?

yes. if the polyclonal is made from the whole peptide (not just a fragment) then it should recognize both. the antibody will contain species against epitopes all over the molecule. some of these will be available when in the native state.

if you are using a commercial antibody then check the information sheet that comes with it. it will tell you with which procedures the antibody will work.

-mdfenko-