Larger proteins can be notoriously difficult to work with when Western blotting. In particular, you can have a hard time achieving a good transfer efficiency. In some ways, large proteins are like teenagers: they are gangly and awkward, stubborn and difficult to get to move in the direction you want them to go.
But give them some love and the proper encouragement, and they will come around.
Try our suggestions for transferring your large proteins out of a gel.
Sacrifice strength for efficiency
Low percentage acrylamide gels can be hard to manipulate. Who wants to try setting up a transfer with a gel that tears as soon as your fingers touch it? But large proteins will move out of a lower percentage gel much more efficiently than out of a higher percentage gel (and they will also resolve better). So increase your dexterity and patience and try a 6-7.5% gel. Or you could try a gradient gel, which resolves both small and large molecular weight proteins.
Dish up the detergent
Larger proteins can precipitate in the gel, inhibiting their transfer. The SDS added during SDS-PAGE usually takes care of this problem, but larger proteins might require more SDS. You can add up to 0.1% SDS in your transfer buffer to discourage precipitation. SDS can inhibit binding of proteins to membranes though, so try starting with a low concentration of SDS (e.g. 0.0375%) and work your way up.
If you do add SDS to your transfer buffer, you many need to re-optimize other transfer conditions such as transfer time and current to prevent the proteins from transferring through the membrane.
Drop the alcohol
Methanol has an opposing effect to SDS. While methanol increases protein binding to membranes, it strips the SDS from proteins. Therefore, reducing the methanol concentration in the transfer buffer will also help prevent protein precipitation. You can reduce the concentration to 10% or less. If you are using a PVDF membrane, you can leave out the methanol altogether. Just don’t forget to activate the membrane with methanol before using it!
Methanol also causes gels to shrink, so if you decrease the methanol your gel will swell more. This will also help when transferring large proteins. But you may want to use filter paper and membrane that is a bit larger than normal to compensate for the larger gel.
Slow down when transferring large proteins
Getting a good transfer efficiency with large proteins might take a little bit more time. It can be hard to move those big molecules around quickly. Try transferring over night at a lower voltage. Just don’t forget to keep your transfer cool!
While semi-dry transfer might be neater to set up and require less buffer, it does have its drawbacks. Semi-dry transfer is generally less efficient than wet transfer and you cannot increase the transfer time to accommodate larger proteins. It’s better to stick with the tank when transferring large proteins.
If you are having trouble coming up with the right transfer buffer composition, why not try buying a transfer buffer optimized for increasing the transfer efficiency of hard to transfer proteins? Advansta’s FLASHBlot transfer buffer is designed for the transfer of high molecular weight and/or low abundance proteins.
If you have any tips for transferring large proteins, share them with us. We would love to hear them!