Western background staining with Primary antibody (Rabbit)

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Tue Aug 19 08:44:11 EST 2008

Am 14.08.2008, 11:01 Uhr, schrieb DK <dk from no.email.thankstospam.net>:

> The best (but more laborous) thing to do:
> Extract cytosolic proteins from the cells (hypotonic lysis/dounce),
> spin in ultracentrifuge to remove all insolubles, concentrate to
> at least 5 mg/ml protein, dialize *extensively* (to remove small
> molecules) against secondary amine-free buffer, couple to
> 1-3 ml of an immobilized support (1:1 mixture of Bio-Rad's AffiGel 10
> and 15 would work best; 5-10 mg/ml of activated gel), pack
> into a small column, then run your antibodies over it (reload
> flow through to be sure). Zero background guaranteed.

However, you may get away with just incubating the cytosol with a membrane  
rather than doing a coupling reaction. That should be a lot quicker and  
cheaper. In this case however I would prefer PVDF over NC because of the  
stronger protein binding.

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