Both beta-actin and beta-tubulin are frequently used as load controls in WB, but can I measure the amount of beta tubulin in my sample and normalized with respect to beta-actin?
In other words, beta-tubulin amounts can still vary in cells depending on the cell type and culture conditions?
Any advice is appreciated. Thank you!
Gloria
Hi Gloria,
If your aim is to measure the amount of beta-tubulin relative to the amount of beta-actin, you need to simply probe the same blot containing all your samples of interest with an antibody specific for anti-beta-tubulin and an antibody specific for beta-actin. You can do this simultaneously as long as you use proven specific antibodies. Use a polyacrylamide gel (10%, 37.5:1) that can give you enough separation between tubulin (52 kDa) and actin (42 kDa), and be prepare to quantify the intensity of the bands after you have developed your blot.
If you are trying to measure absolute levels of beta-actin and beta-tubulin, you will also need purified beta-actin and purified beta-tubulin in order to perform a quantitative western.
Does that help?
One word of warning: beta-actin levels can change in cells so do not assume that they are constant either. You should normalize the amount of protein that you load in each lane using another method such as staining your membrane for total protein.
Reliance on any one "housekeeping protein" is generally inadvisable without careful controls.
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