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
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2 replies to this topic
#2
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Posted 01 June 2009 - 01:09 AM
Why no one reply to me? 
saraarasus, on May 25 2009, 03:19 PM, said:
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
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
#3
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Hmmm, I think it's working
Posted 01 June 2009 - 05:30 PM
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.
