Exposure of Western Blot Films
One of the nice things about western blots is the ability to do a relative quantification between samples. The most common way of doing this is to use computer software for densitometry. Densitometry is a way of measuring the intensity of a band in a gray-scale image.
We will briefly discuss the proper exposure for western blots, and then go through a tutorial for quantifying western blot bands using the NIH ImageJ software. There are other software packages available for western blot densitometry, but ImageJ is easy to use and free. So we will focus on its use here.
Proper Exposure of Films
Proper exposure of films to chemiluminescence is critical for good western blot technique. Films should be underexposed so that we are able to detect differences in protein levels. In other words, we mean that no band should be saturated with signal . As secondary antibodies react with ECL substrates, there will be a period of time when the film darkens linearly. We call this the linear range.
For most commercially available antibodies used with our protocol, the linear range will be somwhere between 5 seconds and one minute.
If a film is overexposed so that the bands darken to a point outside of the linear range, the film will appear as if there are no differences in protein levels, even if there really are differences.
For example, take a membrane with three lanes of Protein X. Lane 1 has the least amount of Protein X, lane 2 has a little more, and lane 3 has the most. If we develop this blot with chemiluminescence and expose it to film for 45 seconds and 5 minutes, it may look something like this:
As a general guide, make sure you can always read text on a printed piece of paper through the bands on your developed film. If you can read the text, the film is underexposed. As an example, use the illustrated film below. You can see the last lane is just barely overexposed.
Ensuring that your films are exposed correctly is the first step toward having a successful, semi-quantitative western blot.
3 Responses to Film Exposure
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1) What is the maximum amount of protein be loaded per well for semi-quantitative western blot?
2) Is there a way to concentrate proteins without affecting the phosphate group of phosphoproteins?
The limit for protein loading is really determined by how the gel runs as you increase the protein mass. As more protein is loaded you’ll start to get poorer resolution. You can probably go as high as 50-60 micrograms before you have a problem (on a mini gel).
For most applications, 20-30 micrograms is ideal. I assume there is some problem with sensitivity?
If you need to concentrate the protein, TCA-precipitation is the most popular. However, you can achieve the same effect if you first immunoprecipitate the total cellular complement of your protein of interest, then western blot for the phospho-modification. If you IP from a 100mm dish, you can get as much as a 10x concentration compared with a regular western blot from a 6-well plate.
To be clear, the membranes (PVDF or nitrocellulsoe) do have binding capacities. But as you increase the amount of loaded protein, you’ll likely run into problems with the gel running before you even come close to reaching the binding capacity of the membrane.