Why GAPDH is used in Western Blot?
1 Answer
GAPDH is often used as a loading control.
Explanation:
In Western blotting we often use GAPDH as a loading control. What this means is that by probing for GAPDH we can check that we have a loaded equivalent amounts of proteins on different lanes of the blot.
An example of use – say we have a disease that we think causes an elevation of a particular protein in the cell. We would make up a sample from "healthy" cells and another sample from "diseased" cells. We would then load equivalent protein amounts of both samples onto a gel for the Western blot.
After probing the blot for our protein of interest we find that in the diseased cells that the protein level is indeed elevated (that is a darker band is produced on the blot). To prove that this increase in protein is due to a change of expression in the cell, instead of a loading artefact (that is, the more instense band is caused by a change in protein expression in the cells, and not by us accidentally loading more of the "disease" sample than the "healthy") we would also probe the blot for GAPDH to show that the total protein levels are the same in both lanes.
Hence, by doing this we are showing that equivalent protein has been loaded in the two lanes and that the change in band intensity we are observing is due to changes in protein expression for the protein of interest.
