hai forum users
i have one problem related with Antigen -antibody interaction

i have been developed antibody against Cry 1Ac insecticidal protein which is frequentally used as a transgene for construction of GM COTTON / TOBACCO . This antibody is working well with purified protein but not in a crude protein of GM COTTON / TOBACCO.
My question is that what may prevent the interaction of Antigen-antibody during ELISA / WESTERN BLOTTING.
plz guide me.


thanking in anticipation.


chandra

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I am assuming you had no signal with crude exttract? if this the case, you may need to add anionic detergent ot you sample buffer (1% SDS or 1% sarcosyl) to ensure complete solubliztion of proteins from you starting sample, otherwise they might sit in the well of the SDS-PAGE gel and never migrate and thus getting no results. Good Luck!

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thanks a lot for proper suggestion, but dear i have been done these all which u are suggesting now. inspite of these all didn;t getting any signal during ELISA. Even when i mixed purified protein with the crude extract of GM cotton/tobacco, didn;t get any signal. so again i am in problem. again hope for proper guidance.
thanx


chandra

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Hi,
I have conciderable experience in developing ELISAs, please If you can provide answeres to these questions this might help trouble shoot your problem better,
What type of ELISA you are doing (direct or indirect, compititve inhibition, sandwich etc...)? What is your coating buffer in the ELISA? do you remove the denaturing detergents from your sample prep before your berform the ELISA? What substrate you are using?

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