Western Blotting

ii. Transfer Step

Like SDS PAGE, the Western Blotting technique involves the use of electrophoresis. In a Western Blot, however, electrophoresis is used as a way to transfer proteins from one medium to another instead of pulling them through a medium to separate them. The proteins that were separated in the SDS-PAGE experiment are transferred from the gel to a nitrocellulose membrane.

The nitrocellulose membrane provides a surface that the protein molecules will stick to. After they are on this support, they are accessible to the antibodies that are used to probe for the presence of a specific protein.

animation of antibodies during visualization

Figure: Animation showing the protein of interest stuck to membrane after transfer. The primary antibody sticks only to the protein of interest and the secondary antibody, which is treated with an enzyme to allow visualization, sticks to the primary antibody.

The transfer process involves the following steps:

Create a "sandwich" comprised of filter paper/the nitrocellulose membrane, and the polyacrylamide gel
Assemble the "blot module", (including the "sandwich" from above) and place it in the buffer tank
Add buffers to the tank, and hook it up to the power supply
Set values on the power supply and run the blot

Creating the Sandwich

The filter paper/membrane/gel sandwich allows the gel and membrane to be placed within the blot module (explained below), and allow current to pass, without the gel or the membrane directly touching the parts of the blot module. The gel and the membrane are flush against each other so that protein molecules will be directly pulled from the gel towards the membrane. Below is a cross section of the filter paper/membrane/gel sandwich with the electrodes (anode and cathode) shown on either side.

image of transfer sandwich

Figure: The sandwich consists of (listed from anode to cathode): filter paper, polyacrylamide gel, nitrocellulose membrane, and another piece of filter paper. The direction of protein transfer is from the gel (anode) to the membrane (cathode).

The steps for creating the filter paper/gel/nitrocellulose sandwich are as follows:

Cut two pieces of filter paper and a piece of nitrocellulose membrane to an appropriate size, and soak them in transfer buffer..
Crack open the plastic cassette, remove one side, and cut away the stacking gel
Place a piece of buffer soaked filter paper on the exposed portion of the gel.
Flip the gel over, remove the other side of the plastic cassette.
Place the buffer soaked nitrocellulose membrane against the exposed gel.
Place the second piece of buffer soaked filter paper on the nitrocellulose membrane (covering it)
Check to see that there are no bubbles between the membrane and the gel. Gently push them out to the side if there are any.

image of membrane from video Video

Figure: Video showing the construction of the filter paper/membrane/gel sandwich, assembly of the blot chamber, and preparation of the electrophoresis apparatus for protein transfer.

Assembly of Blot Module

After the filter paper/gel/membrane sandwich has been assembled, it should be placed in the blot module. The blot module contains two flat electrodes and uses spongy pads to hold the gel/membrane/filter paper sandwich in the proper orientation between these electrodes during electrophoresis.

Shown below are the two halves (i.e. the cathode half, and the anode half) of the blot module, with blotting pads in place. Remember that you would like negatively charged protein molecules (residing in the SDS PAGE gel) to travel toward the membrane when current is applied.

image of blot module setup

Figure: The blot chamber with (from negative to positive): two blot pads, filter paper, gel, membrane, filter paper, and two more blot pads.

After the blot module has been assembled, it is placed in the buffer tank. A wedge is used to press the two halves of the blot module together and hold it in place in the tank.

Figure: Electrophoresis apparatus with the blot chamber inserted and a wedge (clamp) to seal the chamber and hold it in place.

Running the Blot (Electrophoresis)

To run the blot, final assembly of the electrophoresis apparatus is necessary, and then the voltage is set and applied for a period of time.

top view of prepared electrophoresis apparatus

Figure: The blot chamber(interior) filled to a level above the top of the filter paper/membrane/gel sandwich with transfer buffer and the lower chamber (exterior) filled with either water or transfer buffer.

Specifically, the interior of the blot module is filled with transfer buffer, and the exterior area is filled with water or transfer buffer. The lid is placed on the tank and then the leads from the lid are plugged into the terminals in the power supply.

Figure: Electrophoresis apparatus with the blot chamber and top in place and attached to the power supply.

Upon verifying that the connections are correct, the voltage should be set on the power supply. Optimal run conditions are usually determined experimentally. In this case a voltage of 30 volts for one hour is suitable. After the run is completed the power supply is disconnected, the lid is removed, and the blot module is taken out of the tank and disassembled.

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