Western blotting

Following electrophoresis, proteins in a polyacrylamide gel can be transferred to a positively charged membrane (e.g., Schleicher and Schuell BA85) in a buffer-tank–blotting apparatus or by semi-dry electroblotting as described below.

With the semi-dry electroblotting method, the gel and membrane are sandwiched between two stacks of filter paper that have been pre-wet with transfer buffer. The membrane is placed near the anode (positively charged), and the gel is placed near the cathode (negatively charged). SDS-coated, negatively charged proteins are transferred to the membrane when an electric current is applied. With the tank-blotting method, a blotting cassette is submerged in a tank for blotting (see figure Tank- and semi-dry blotting methods).Tank blotting can be performed over extended periods since the buffer capacity is far greater than that with semi-dry transfer systems. Results obtained with the tank-blotting method are typically better, with more efficient transfer, particularly of large proteins. Transfer efficiency can be checked by staining proteins on the membrane using Ponceau S (see Ponceau S staining). Once transferred to the membrane, the proteins can be probed with epitope-specific antibodies or conjugates.

Materials required

• Transfer apparatus
• Filter paper (e.g., Whatman 3MM)
• Positively-charged membrane (e.g., Schleicher and Schuell BA85)
• SDS polyacrylamide gel containing separated proteins (see Separation of proteins by SDS-PAGE)
• Transfer buffer (semi-dry or tank-blotting) (see table Semi-dry transfer buffer or Tank-blotting transfer buffer)

Semi-dry transfer buffer

Tank-blotting transfer buffer

1. Cut 8 pieces of filter paper and a piece of membrane to the same size as the gel.
   
   Tip: To avoid contamination, always handle the filter paper, membrane, and gel with gloves.
   
   
2. Incubate membrane for 10 min in semi-dry or tank-blotting transfer buffer.
3. Soak filter paper in semi-dry or tank-blotting transfer buffer. Proceed to step 4 if performing semi-dry transfer or step 5 if performing tank blotting.
4. Semi-dry transfer: Avoiding air bubbles, place 4 sheets of filter paper on the cathode (negative, usually black), followed by the gel, the membrane, 4 sheets of filter paper, and finally the anode (positive, usually red).
5. Tank-blotting: Avoiding air bubbles, place 4 sheets of filter paper on the fiber pad, followed by the gel, the membrane, 4 sheets of filter paper, and finally the second fiber pad.
   
   Tip: Air bubbles may cause localized nontransfer of proteins. They can be removed by gently rolling a Pasteur pipet over each layer in the sandwich.
   
   
6. Carry out the transfer procedure. For current, voltage, and transfer times specific to your apparatus, consult the manufacturer’s instructions.
   
   Tip: Time of transfer is dependent on the size of the proteins, percentage acrylamide, and gel thickness. Transfer efficiency should be monitored by staining (see below). The field strength required is determined by the surface area and thickness of the gel: 0.8 mA/cm2 is a useful guide (1 h transfer).
   
   
7. After transfer, mark the orientation of the gel on the membrane.

1. Incubate membrane in Ponceau S staining solution (see table Ponceau S staining solution) with gentle agitation for 2 min.
2. Destain in distilled water until bands are visible
3. Check that proteins of different sizes have been transferred uniformly to the membrane. Hydrophobic proteins may be more efficiently transferred by increasing the percentage of methanol in the transfer buffer.
4. Mark membrane using a suitable pen (i.e., one not containing water-soluble ink) or pencil, or cut as desired.

Ponceau S staining solution