With a western blot, scientists in a research laboratory can identify what protein is in a given sample, as well as how much is present. It’s an ideal way to find out the expression of protein levels over time, for example, or to monitor how protein expression may be changed in a sample from a patient. You use western blotting to identify a disease in a lab setting or when testing the efficacy of a new therapy.
A major concern among technicians is how accurate the data is when doing a western blot. It’s key to make certain that any observable differences in your data are caused by changed protein expression levels and not protein transfer or gel loading mistakes. So, in tests and experiments, you will employ western blotting loading controls. For this, you require housekeeping proteins.
Note that the term “western blot” is not capitalized. The name is a bit of a play on words and is based on a forerunner of RNA molecule identification called the “northern blot,” which was named to follow the first version of the test, a “southern blot” invented in 1975 by Edwin Southern.
Using Loading Controls in Western Blotting
Loading controls are antibodies used to determine the identity of a protein expressed in a particular kind of sample or cell. Housekeeping genes encode housekeeping proteins. They are needed in the cell to regulate and maintain its basic functions.
You want to employ loading controls when your experiment calls for comparing the signal strength of different proteins, from sample to sample. And if you are preparing to publish a scientific paper on your research, journals will require that you employ controls that are appropriate to the experiment you’re conducting, per Bio-Rad.
About Housekeeping Proteins
Working with western blotting tests, researchers must make sure that the changes they are monitoring in a sample under testing are not the result of loading inconsistencies or because a protein is missing. This is where housekeeping genes come into use.
Housekeeping genes express proteins that work to maintain basic functions in a cell. Scientists work under the assumption that housekeeping genes are not subject to many alterations in their expression pattern, as noted by Advansta.
Examples of housekeeping genes that are used during western blotting tests include “glyceraldehyde 3-phosphate dehydrogenase (GADPH), beta-actin, tubulin or heat shock protein 90 (HSP90).”
Housekeeping genes typically express regularly, with their expression patterns not altering in most cases. But sometimes, they are influenced by a particular treatment protocol.
For example, beta-actin expression is affected by spinal cord injuries, but beta-tubulin is not affected in this way. And when scientists are working on an experiment that involves using infectious viruses to deliver exogenous genes, the housekeeping genes can be changed during the process.
This means that scientists and researchers working with western blotting will prefer to use housekeeping genes that offer them the strongest possible signal in their experiments. What’s called for is to find a housekeeping gene that has an expression level matching the protein you are studying.
Selecting Housekeeping Proteins for Western Blotting Research
There are a lot of factors to consider when doing western blotting tests, with scientists needing a high level of precision to see how their protein expression levels are changing during experiments.
So housekeeping proteins help to sort out differences caused by altered protein expression or differences stemming more from errors of protein transfer or the gel loading process. The trick is in doing research to determine the most appropriate housekeeping proteins for your particular experiment.