You look at the results of your Western blot and you can tell that the levels of your protein are changing. You can probably even guess that it is about a 5 or 10 fold change. But how will you really know?
Many people turn to quantitative Western blots to better describe differing amounts of protein. However, if not performed correctly, these assays can give pretty erroneous results.
Make sure you are accurately quantifying Westerns by following our guide to Important Factors in Performing Quantitative Westerns.
And by watching out for the common mistakes listed below:
Using x-ray film
The absolute best way to quantitate a Western is by using an imager to capture the signal (such as a charge-coupled device [CCD] camera system). To your naked eye it may appear that you can capture a wide range of protein concentrations on film, but it just ain’t so. Weak signals are difficult to capture because they require long exposure times that also invariably increase the background signal. Strong signals are equally troublesome as they max out the capability of the film and are no longer within a linear range in which the signal is directly proportional to the protein concentration.
Not titrating your antibody
Yeah, it can be a pain to do an experiment just to optimize the amount of antibody to be used in subsequent experiments, but it is well worth the time investment. A well-titrated antibody will increase the sensitivity and thereby decrease your limit of detection, increase your signal to noise ratio and broaden the linear dynamic range.
Choosing a bad internal reference (or not using one at all)
Measurements of your target protein amount should be normalized to a protein whose concentration doesn’t change from sample to sample. Normalization to a standard reference factors out inconsistencies in loading of samples (oops, did I just bump the tank?) and variances in total protein concentrations from sample to sample (yes, ethanol does kill off some of the cells). But it only works if the standard is expressed consistently between samples.
Housekeeping genes (such as GAPDH or beta-actin) are expressed constitutively and their protein products are required for basic cellular function, therefore they are often chosen as good internal controls. However, make sure that none of your experimental conditions alter their expression before you use them for comparison.
Alternatively, you could try using a total protein stain.
Comparing between blots
No matter how much you try to replicate experiments, things change from day-to-day….you have to make new transfer buffer, the undergrad breaks your favorite transfer apparatus, your PI interrupts you while your blot is incubating in substrate… Even 2 blots run on the same day will experience different variations in transfer efficiency. If you want to accurately report the effect of different treatments or conditions on protein amounts, then they must all be run on the same blot.
Not being careful
Western blotting is, by its very nature, an imprecise technique. There are many things that cannot be controlled – make sure you are careful with the things that are in your control. Follow a protocol carefully. Be careful loading your samples, standardize your incubation times and be careful when you make up your buffers.
The more precise you are, the more accurate your measurement will be.
Photo courtesy of Kevin Dooley.
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