b'Pre-transfer considerationsChoice of transfer system Building the western transfer sandwichWhen choosing a transfer methodology, convenience,After proteins have been separated by electrophoresis, speed, flexibility, and throughput are importantthe next step of western blotting is the assembly of the considerations. While the devices the laboratory currentlytransfer sandwich. For wet and semi-dry electrotransfer has may be the most convenient, consider how alternativedevices, the transfer sandwich typically consists of a filter methods may improve steps such as transfer preparationpapergelmembranefilter paper arrangement, where the and cleanup. A dry transfer system, with ready-to-usefilter paper aids in wicking of the transfer buffer. For some consumables, provides greater convenience, requiringcommercially available wet and semi-dry transfer devices, very little setup and cleanup time. Regarding transferpre-assembled transfer stacks are available with pre-cut speed, wet transfer systems are typically the slowest,membrane and filter paper, in which the polyacrylamide gel followed by semi-dry transfer (using Towbin buffer), withis inserted. dry transfer systems being the fastest. However, with the specially designed transfer buffers for semi-dry systems,Dry transfer devices do not require filter paper for wicking semi-dry systems can match the speeds offered by drytransfer buffer. Instead a unique gel matrix transfer stack transfer systems. Wet tank systems come in a variety ofincorporates buffer, eliminating the need for buffer tanks capacities and designs and typically offer the greatestand wetted filter paper. flexibility in throughput, but this attribute should be weighed against the transfer speed. One may be able toChoosing the western blot membranecomplete the same number of transfers (or more) usingThe most common immobilization membranes for western a fast, lower-throughput, semi-dry or dry transfer systemblotting are nitrocellulose, PVDF, and nylon. The following compared to using a slower wet tank transfer system.characteristics make these membranes suitable for protein transfer: With respect to transfer efficiency, most modern transfer systems will transfer typical protein molecular weight A large surface area:volume ratioranges with high efficiency. Most modern semi-dry systems A high binding capacityand dry systems offer preprogrammed optimized methods for particular molecular weight ranges, including high Extended storage of immobilized proteinsand low molecular weights. Transferring very high or very Ease of uselow molecular weight proteins often requires optimization regardless of the system used. However, because of the Possible optimization for low background, signal,flexibility of wet transfer (for example, transfer membranesand reproducibilityof different pore sizes can be swapped easily and transfer buffer formulations can be modified), wet tank transfer isWestern blot membranes are typically supplied in either a good place to start when transferring very high or lowsheets or rolls, and commonly have a thickness of 100 m, molecular weight proteins. See Table 1 on page 6 for awith typical pore sizes of 0.1, 0.2, or 0.45 m. Most proteins summary of Invitrogen transfer system characteristics. can be successfully blotted using a membrane with a pore size of 0.45 m, while low molecular weight proteins or peptides (20 kDa) usually require a pore size of 0.1 or 0.2 m. For some transfer instruments, pre-assembled membrane and blotting paper stacks are available.Learn more atthermofisher.com/transfer16'