Transfer membranes of different pore sizes can be swapped easily, and transfer buffer formulations can be modified. Gels are still compatible with downstream immunodetection. First, proteins are separated from each other based on their size by SDS-PAGE gel electrophoresis. When performing traditional wet tank transfers and traditional semi-dry transfers, optimal results are obtained when the electrophoresis buffer saturating the gel is replaced with transfer buffer. It also provides some troubleshooting tips for electrophoretic transfer and outlines a general workflow for the protein transfer from gels to membranes. Transferring protein from gel to membrane.
But alcohol may cause a reduction in the gel pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. However, alcohol in the transfer buffer improves protein binding to nitrocellulose membranes by stripping the SDS from the proteins and increasing hydrophobic interactions with the membrane. Note: GAM = goat anti-mouse, HRP = horse radish peroxidase, TBST = tris-buffered saline with Tween (solution), GAR = goat anti-rabbit.
When using the Power Blotter Semi-dry System, proteins >150 kDa migrate more slowly and require more time to transfer. Tris-acetate gels afford the best separation of HMW proteins.
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