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antribody gone old? - (Jul/08/2009 )

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thanks for replies.

Does anyone know sensitive proteins are to heat during transfer? Is 40 degrees allready too much?


are you running a Native or a SDS-PAGE? If it's SDS-PAGE, I think you should have SDS in your transfer buffer. Over eating during transfer is problematic. One of the reasons i think is I alters your gel (it kind of cooks it) and some protein might remain stuck in it. I doubt 40 C is problematic. Anyway for SDS-PAGE, you proteins are already denatured. As for a Native, well 40 C is just 3 C above normal body temp, so a vast majority of protein should still have their original conformation.
Are you using PVDF or nitrocellulose? If your are using nitrocellulose and your transfer is too long, your proteins can be pulled through and out of you membrane.

anyway, good luck

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