Semi-Dry transfer large protein - (Aug/28/2008 )
I'm working with a large protein (Molecular Weight of 2 isoforms: 395/345 kDa). I use a low percentage acrylamide gel (5%, 1.5 mm) and I want to transfer proteins on PVDF membranes (Buffer with 10% Methanol).
I use "Trans-Blot semi-dry transfer cell" (BioRad).
I realized a transfer at 10V during 1 hour, but it's not satisfactory. Please, what parameters could be used for semi-dry transfer for alrge proteins ?
Any ideas would be appreciated, Thank you.
I am not sure but I have read somewhere, that the bigger your protein is the longer and slower you should transfer it. So, normally you would use 2-2,5 mA/cm², I would start and try with 1mA/cm² for the double time and see, what result you will get. At 4°C, as usual.
I include 0.05% SDS in my transfer buffer to transfer a 300 kDa protein out of a 10% gel at 30 V for 2 hours which works nicely using a semi-dry blotter
My protein is 30 kD. I used 200 mA for 30 mins for every thing. If you have the prestained protein marker, you can see the marker on the membrane after the transferring.