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Information on transferring proteins to membranes.
Western Blot Transfer
Western Blot Transfering Background Western blot is in fact a method which involves blotting hence the term "blot". Blotting involves the transfer of proteins from the gel matrix to the membrane. This allows the proteins to be placed on a membrane and be amenable (accessible) to detection using antibodies. The modern western blot protocol involves transferring proteins in the presence of electrical current, usually in a transfer apparatus. Types of Transfer Membranes in Western Blot Western blot employs several popular membranes. These included nylon which is not commonly used anymore, nitrocellulose and PVDF. See Benefits of Membrane Types in Western Blot To summarize, membranes are chosen for their non-specific binding properties of proteins in that membranes bind all proteins equally well and not certain proteins more than others. The binding of proteins to membranes is based mostly on hydrophobic and charged interactions between the membrane and proteins. In general, nitrocellulose membranes are much cheaper than PVDF membranes. However nitrocellulose membranes are far more fragile and do not stand up well to repeated stripping and reprobe cycles. Western Blot Transfer Method The western blot transfer method as mentioned used capillary action previously. In this older method, the membrane was placed on the top of gel and water-absorbent tissue papers were placed on top. The entire pile was placed in transfer buffer solution which moved up the paper by capillary action. The capillary movement of fluid to the membrane would transfer the proteins to the membrane. These days, transfering is done in a transfer apparatus in an electrical current, usually 35 A. This is transfer of proteins to a membrane in electrical current has been called electroblotting. Electroblotting Electroblotting is a transfer method which takes advantage of the fact that SDS negatively charges proteins. In an electrical current, these proteins will move and hence can be transferred efficiently in an electrical current to a membrane. In summary, transfer of proteins to a membrane simply "transfers" proteins to structure which not only maintains the order and distribution of proteins that was in the gel (hence allowing quantitative analysis), but it also allows proteins to be exposed to antibody detection on a thin surface layer. Analyzing Transfer Methods Transfer of proteins can be analyzed for their uniformity and overall effectiveness of transfer by various protein staining methods. For example, membranes may be stained with Coomassie or Ponceau S dyes. Coomassie is more sensitive, although Ponceau S's water solubility does make it easier to subsequently destain proteins and probe the membrane.
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