Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: separation by size, transfer to a solid support and marking target protein using a proper primary and secondary antibody to visualize.
To prepare the resolving gel (10 ml) and stacking gel (3 ml), different concentrations and volumes can be used as per the purpose.
- distilled water
- 30 % Acrylamide mix
- 1.5 M Tris (pH 8.8)
- 10 % SDS
- 10 % APS
Gel is cast between two glass plates, deemed the short plate and spacer plate, with 1 mm or 1.5 mm width. Prepare the resolving gel first and use 10 µl to speed up polymerization.
The plates are inserted in the casting frame and the clips are closed such that the plates are sealed and leveled.
The prepared resolving gel is poured between the glass plates using a 1 ml pipette. Butanol is added afterwards to smooth the gel and remove bubbles.
After the resolving gel has solidified, TEMED is added to the stacking gel and it is added on top of the resolving gel to the upper edge of the plate. The comb is then immediately added.
The glass plates are removed from the stand and inserted into the running chamber holder with the buffer dam on the opposite side. Running buffer is added between them and the holder is then transferred into the running apparatus. The wells are covered with running buffer and the samples are loaded very meticulously.
Samples are loaded from right to left so that after the transfer, they appear from left to right. Loading volume should be the same for all samples and the sample buffer is loaded after the last sample. The gel is run for 2 hours at 100 volts.
Afterwards, the transfer is prepared for the gel. A sandwich is prepared in the following order: black side, sponge paper, filter paper, gel (flipped), PVDF membrane, filter paper, sponge pad and white side of the sandwich.
After the transfer is complete, the membrane is blocked to get rid of any non-specific signals. Blocking buffer is prepared with 1 gram of milk powder and 20 ml of TBST. After this, there are primary and secondary antibody dilutions on the rocker pictured below.
A substrate is prepared. This is detected by the machine and helps produce images when developing the blot. Stripping is done afterwards to remove previously probed proteins in order to probe for other proteins.
This is a fairly common procedure used in many labs today. The procedure is very long but worthwhile in the end.
Author taught specific procedure during time working in a lab.