Basics

• Following electrophoresis, the polyacrylamide gel is removed from the buffer tank and the proteins it contains are transferred to a nitrocellulose or PVDF membrane. The most common method for transferring proteins is the semi-dry method. In this approach, a filter paper and membrane are placed on the anode -- a positively charged electrode. Another piece of filter paper is placed atop the gel, followed by the cathode. The proteins have already been denatured and are coated with a compound called sodium dodecyl sulfate, so they all have negative charge. Consequently, the voltage drop between the two electrodes draws the proteins out of the gel and into the membrane. Several factors affect the effectiveness of this transfer.
  
  

Current

• Although voltage and current are of course related by Ohm's Law (V = IR), current is a more important than voltage in this case. If the current is too high, it could cause overheating. Moreover, a high current could also cause smaller proteins to move so quickly they don't have a chance to bind to the membrane. Choose a buffer that has a lower charge density to prevent the current from becoming too high; Tris-Glycine is a good example. Also make sure to dab up any excess buffer lying around the filter paper/gel stack. Pools or splashes of buffer could connect the electrodes directly, creating a short.
  
  

Membrane

• You can buy membranes with different pore sizes; the size you choose depends on the size of the proteins you want to study. 0.45 micrometers is a common choice, although if the molecular weight of your protein is below 20 kiloDaltons, you may need a membrane with an even smaller pore size. Also choose carefully when deciding what kind of membrane you want. PVDF is more expensive than nitrocellulose, but it's less fragile and you can probe it repeatedly, whereas nitrocellulose membranes can typically only be probed once.
  
  

Buffer Chemicals

• Including methanol in your buffer is important, because it helps to remove SDS from your proteins so they can bind to the membrane. Adding too much methanol, however, removes the SDS too soon, before the proteins have left the gel, which could cause them to clump up and become trapped in the gel. Depending on the type of experiment and protocol, 20 percent methanol is a reasonable concentration, although it's important to include a low concentration of SDS as well. This additional SDS helps the proteins to elute, or be extracted, from the gel.
  
  

Protein Charge and Other Factors

• As the methanol dissolves the SDS, the proteins become less negatively charged. In some situations this can pose a potential problem, because the proteins migrate more slowly as their net charge becomes less negative. If you find your proteins will not properly transfer for this reason, you can try a buffer with a higher pH, like CAPSO (3-N-cyclohexylamino-2-hydroxypropane sulfonic acid), since your proteins will have a more negative charge at the higher pH. Another factor to consider is the time of transfer; larger proteins move more slowly, so you need to allow sufficient time for your proteins to transfer to the membrane.