b'TransferI ntroduction to transfer methods for western blottingProtein transfer from gel to membrane is necessaryAnode core (+)for two reasons: better handling capability offeredSponge padby the membrane than the fragile gel during westernFilter paperblot processing, and better target accessibility onTransfer membraneGelthe membrane by macromolecules like antibodies.Filter paperIn general, all electrotransfer methods rely on theSponge padelectrophoretic mobility of proteins to move them out of aCathode core ()gel. The techniques involve placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose or polyvinylidene difluoride (PVDF) membrane or other suitable protein-binding support. Next, the gel and membrane are sandwiched between two electrodes, which are typically submerged in a conducting solution (transfer buffer) (Figure 1). When an electric field is applied, the proteins move out of the gel and become tightly attached on the surface of the membrane. The Figure 1. Western blot electrotransfer of proteins from gel to resulting membrane is a copy of the protein pattern thatmembrane. While this diagram depicts the setup of a typical wet transfer, Direction of transferwas in the gel. For a complete workflow, see Figure 2.many of the principles apply to semi-dry and dry methods of protein Prior to electrotransfer, the most popular method for proteintransfer to membranes.transfer was diffusion blotting, which took an average of 1.5 to 2 days. In comparison, a typical dry electrotransfer Prepare transfer bu\x13ernow averages less than 10 minutes. In this handbook, we will only be discussing electrotransfer and will refer to thisPrepare transfer bu\x13er su\x12cientfor the transfer tank andas transfer or blotting. for equilibration of gels and membranes.There are three ways to transfer proteins from SDS-PAGE Equilibrate gels and membranesor native gels to nitrocellulose or PVDF membranes for the purpose of western blotting: wet transfer (also knownEquilibrate gels and membranesin transfer bu\x13er. as tank transfer), semi-dry transfer, and dry transfer. Support gridThis handbook will focus on these three techniques andPadsAssemble the gel and membrane sandwich Filter paperother considerations to help improve your protein transferTransfer membraneefficiency for better western blot results. Place the membrane and gel Gelbetween bu\x13er-soaked lter papers.Set up the transfer cellPlace the gel, membrane, and lter paper GelmembranelterDid you know sandwich in the transfer tank. paper sandwichFill the tank with transfer bu\x13er.W. Neal Burnette in Tobert Nowinskis lab at the FredConnect the tank to the power supplyHutchinson Cancer Research Center in Seattle was the firstand set the power supply Buer tankto coin the term western blotting. The term honors Edwinfor optimal power and time. Southern, who described blotting of DNA, and is a referenceAnode (+)to the West Coast location of Nowinskis lab.Cathode () ElectrodesStart the transfer Direction oftransferFigure 2. Workflow of the tank electrotransfer of proteins for western blotting.5'