Do you have experience with diffusion blotting from SDS-PAGE? Would you mind sharing a protocol? Prof. Ingrid Olsen shared a great protocol for precast 0.5mm GE Healthcare SDS-PAGE gels in the book Methods in Molecular Biology - Western Blot methods and protocols. I am wondering if anybody has a protocol for diffusion blotting of self made SDS-PAGE gels? I am wondering if my gel would break under pressure of 6g/cm2.
Best,
Jan
Use the same procedure that works for precast gels (at the same or similar acrylamide concentration). If you are using lower concentration of acrylamide than your precast gels where the transfer procedure worked successfully, reduce the weight enough so gels are not getting deformed.
Best,
Hi Jan,
as we are using film backed precast gels for 1D and 2D PAGE gels, we started using dry blotting (Beo Dry Blotter) as we did not wanted to peel of the gels from the film (0,5 mm gels brake very easily). For special approaches we designed a special blotting tool with which we can control the pressing force precisely (e.g. EPO diagnostics). Maybe this blotting tool could work for you too (Velum Blotter).
Best,
Kristin
P.S. Multiple blotting from one gel as done in the paper you mentioned is possible the Beo and Velum Blotter as well.
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