Crosslinking of oligos to membrane required for EMSA? - Is it necessary to crosslink the oligos to a positively charged nylon (Jul/17/2007 )
We're setting up non-radioactive EMSA with DIG-labelled oligos.
Is it necessary to crosslink the oligos to a positively charged nylon mebrane when performing EMSA? If so, what is the best way? I read about baking 1-2 h at 80C, baking 2-3 min in microwave, UV irradiation.
Cheers, Leon.
-LJSB-
I tried a non-radioactive EMSA the other week with Biotinylated oligos. I transfer in 0.5X TBE in a wet western transfer mini transblot apparatus onto Hybond N membrane and forgot to UV cross link. It worked fine. The persons protocol I was following said to UV cross-link on a transilluminator for 15mins.
-Ceri-
QUOTE (Ceri @ Jul 18 2007, 10:39 AM)
I tried a non-radioactive EMSA the other week with Biotinylated oligos. I transfer in 0.5X TBE in a wet western transfer mini transblot apparatus onto Hybond N membrane and forgot to UV cross link. It worked fine. The persons protocol I was following said to UV cross-link on a transilluminator for 15mins.
Thanks for your reply. What conditions (time, current/voltage) do you use for the wet transfer?
It seems that the mode of transfer matters. Somewhere I read that cross-linking is not necessary after alkaline transfer.
Can someone confirm this?
Cheers, Leon.
-LJSB-