All of that is necessary, but it’s also tedious and time consuming. Positive and negative controls must be separately established, consistent protein expression levels across different samples should be tested, and a scalar linear signal response needs to be confirmed across a range of protein loading amounts ( Janes 2015). Oh cautions that there are constraints to using single protein detection. Luckily, thanks to the nature of housekeeping proteins, antibodies for them are readily available and detecting ubiquitous proteins is easy.īut, Dr. That is to say your housekeeping protein will not be your target, but it should still be ubiquitous and consistently expressed in your sample. The key to success with this approach is to base normalization on a protein that can take on the role of being an internal standard. Their abundance within a sample serves as a proxy for the entire protein population. There are two approaches to normalization when evaluating a western blot: single protein detection and total protein normalization.īy far the most common approach to single protein detection is to use housekeeping proteins. Regardless of which method you ultimately decide to use, the criteria for any good normalization technique is to establish a region of linear signal response: quite simply, the increase in signal is directly and linearly proportional to the amount of protein contained within the sample. Each technique has its own set of considerations. When comparing any given data point to a whole set, you’ll need to know what the whole set looks like, mathematically. There are multiple ways to normalize your western blotting data and in theory, they should provide you with the same desired result: the amount of a specific protein or specific proteins contained within a sample, relative to the other samples run in the same blot. Normalization is the analytical tool that allows you to accurately compare how various data points within a specific set of data are different, relative to each other. You may be asking yourself if normalization in western blots is the same thing as the data normalization you learned about in Stats 101.
#WESTERN BLOT QUANTIFICATION USING IMAGEJ SERIES#
He launched the opening chapter of the western world webinar series by introducing the mathematical technique every wise scientist needs to perform to report meaningful data from their western blots - normalization. Oh, our very own in-house western blotting guru, kicked off a webinar series covering everything you could want to know about western blotting (which you can find at the end of this article).
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