Immunization with protein band from SDS-PAGE - (Apr/17/2008 )

Hello,

I need to inject mice with a protein band excised from SDS-PAGE gel. Can someone please tell me the procedure for this. Is it necessary to use freund's adjuvant while using this protocol, won't the acrylamide itself act as an immunogen?

Thanks in advance.

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Protocol I have but haven't used says you have to stain your gel, fix with 2% glutardehyde and destain, then cut your band of interest, pulverize in an Eppendorf tube, freeze-dry (lyophilize), mix with PBS and adjuvant, emulsify and inject.

Personally I would fix first, wash to remove glutardehyde, stain with reversed staining (eg. zinc, CuCl2) and then cut.

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we used stained gel bands, cut out, homogenized, mixed with adjuvant, and injected.

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Freund´s adjuvant is helpful, I think acrylamide itself works as a mild adjuvant; booster after 6 weeks

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I am rather new to this. How do u estimate how many bands you need to cut out to inject in a mouse? i am expressing this protein ibn E.coli and the expression is quite high.

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we made preparative gels (we loaded close to mg quantities) and stained with coomassie. we assumed that there was enough for a few injections (initial and boosters) but we could prepare more, if necessary.

even with analytical gels, if you have enough to see with coomassie then you should have enough to inoculate (especially if you ran multiple lanes, it also depends on whether you are injecting rabbits or mice).

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