Casp3 - (May/25/2009 )
Hi,
I am using the cell signaling casp3 with PVDF. I have a strange problem. My protein is transfering so fast. I am using wet transfer system, 100 volt, 0.23 amp and transfer it for 30 min. I think most of the protein has just passed. My transfer buffer doesnt have SDS , but it has 20% Meoh to help them to attached better to the membrane. I am not really sure which volt or current I use to transfer them good in 1 hour. I would be really appreciated if some one can help.
Cheers,
Sara
Why no one reply to me? 
saraarasus on May 25 2009, 03:19 PM said:
I am using the cell signaling casp3 with PVDF. I have a strange problem. My protein is transfering so fast. I am using wet transfer system, 100 volt, 0.23 amp and transfer it for 30 min. I think most of the protein has just passed. My transfer buffer doesnt have SDS , but it has 20% Meoh to help them to attached better to the membrane. I am not really sure which volt or current I use to transfer them good in 1 hour. I would be really appreciated if some one can help.
Cheers,
Sara
No-one replied as you didn't actually state the problem, which I guess is that you have no signal from your caspase 3 blot? I would try transferring slower - use 15 V overnight and use SDS in your buffer. The SDS actually coats the protein with a negative charge and allows it to migrate.