I am facing problem in the transfer of high molecular weight proteins to a nitrocellulose membrane from SDS-PAGE gel. Experimental target protein has a molecular mass of ~125kDa, and I tried different concentration of gels. I try both wet and semi-dry procedure but never achieve complete transfer of proteins. What will be the possible solution for this problem?
I would recommend performing the transfer overnight at 4 degrees at 15 volts. I have had better success using a wet transfer for proteins that size, but semi wet works as well. I agree with the above posters in that it would be helpful if you listed what type of transfer buffer you have.
I am not sure what kind of transfer buffer you are using and what exactly you have already tried. One solution is to increase the time of transfer up to1.5 to 2 hours. I, on the other hand, decided to save myself a hassle and I invested in 3-8% Tris-acetate precasted gels from Life Technology. The system works amazing, I am able to get the perfect transfer of proteins with MW between 120 -300kDa.
Hi, it would be easier to give a hint, if we know which buffer systems you are using/- time and mAmpere-conditions. Furthermore have you the possiblity to use PVDF-Membran instead of Nitrocellulose? AtB
I would recommend performing the transfer overnight at 4 degrees at 15 volts. I have had better success using a wet transfer for proteins that size, but semi wet works as well. I agree with the above posters in that it would be helpful if you listed what type of transfer buffer you have.
Hi,
Mobility (within the gel and outside of it) improved a lot when working with lower total acrylamide gels, as most colleagues have pointed out. 6% would be awesome if it wasn't so difficult to manipulate later... 7.5% was a happy medium for me. But, what worked wonders was the thickness of the gel: try 1.5mm thick gels... big proteins appear to have more room to move there :) I supplemented in the transfer buffer with 0.05% Tween-20 rather than SDS, to use a less chaotropic agent. Finally, I transferred overnight (16 hours) at 20V at 4ºC.
All of the above: low percentage gel, longer transfer (overnight works well), single blot transfer, central lanes
PLUS: prefer wet transfers, fresh transfer buffer....least re-add Methanol.
Best wishes and good luck!
Thanks to all for their valuable information contribution. I tried 8%, 9%, 10% and 12% gels. Transfer also overnight at 35V at 4ºC. I used transfer buffer having constituents (25mM Tris Base, 192mM Glycine, 10%/20% methanol/ 25mM Tris Base, 192mM Glycine, 10%/20% methanol, 0.1% SDS) in wet transfer. But still not achieve any good transfer.
I work with some proteins between 100-250 kda, 8%gels and transfer them at 120v during 70 min usually is sufficient. Transfer buffer: 14.4 g glycine, 3.03 tris, 200ml methanol to prepare 1lt
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