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Western Blot Optimization:
Protein Transfer
The biggest differences between western blot protein transfer types arise in the type of equipment needed and the speed and efficiency of the transfer. Pick your transfer method carefully to get the best results in your own time and for the right price. Learn more about protein transfer optimization here.
How to transfer Large proteins for Western Blotting
Tips on how to optimize the transfer of large proteins.
Larger proteins can be difficult to work with when especially on Western blotting protein transfers. Find some tips here on how to deal with transfers of large proteins during western blotting.
Low percentage acrylamide gels can be hard to manipulate. However, 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).
There is a probability of Larger proteins precipitating in the gel, inhibiting their transfer. If you do add SDS to your transfer buffer, you may need to re-optimize other transfer conditions such as transfer time and current to prevent the proteins from transferring through the membrane.
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.
The 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.
How to Select the Right Transfer Type
Transferring electrophoretically separated proteins to a membrane allows easier handling and better accessibility to antibodies, stains, and signal development reagents. All transfer types rely on the electrophoretic mobility of proteins to push them out of the gel and onto the membrane; the biggest differences between transfer types arise in the type of equipment needed and the speed and efficiency of the transfer. Pick your transfer method carefully to get the best results in your own time and for the right price.
Wet Transfer
Wet transfer is the most common protein transfer method. After assembling the transfer sandwich with saturated
filter paper, the assembly is locked into a transfer cassette and immersed in a transfer tank filled
with buffer before the electric field is activated. This type of transfer is usually run for several
hours or even overnight.
Wet transfers are more consistent than faster methods, allowing more quantitative interpretation, but are
slow and prone to errors caused by bubbles trapped between the layers of the sandwich.
Semy-dry Transfer
Semi-dry transfer sandwiches are of the same basic setup as for wet transfer, but rather than immersion in the buffer, the components of the sandwich are soaked in the buffer and held between transfer pads and electrode layers. The semi-dry transfer is much faster than wet transfer, typically taking only about 7 minutes. The rapidity of the transfer can cause uneven, blotchy, and inconsistent results. This method is more expensive due to the specialized equipment and consumable materials required.
Dry Transfer
Dry transfers use a unique gel matrix in place of buffer tanks or soaked filter paper. The high ionic density of the gel matrix enables more rapid protein transfer. Since this method does not involve immersing the electrodes in liquid, no oxygen is produced during electrotransfer, which reduces blot distortion. Dry transfer is fast and produces more consistent results, but is also the most expensive option.
Keywords: Western blotting optimization, protein transfer, wet transfer, semi-dry transfer, dry transfer, best transfer method
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