What’s your quantitative western blotting IQ?
Are you doing everything you should to ensure accurate quantitative Western blot data? Find out by testing yourself with this quantitative western blotting quiz. If you get one or more questions wrong, you can brush up on the basics by downloading our Quantitative Western Blotting Basics guidebook using the form on the right.
1. True or False: To get quantitative western blotting data do the following:
- Follow your typical western blotting protocol. Be sure to probe for your protein-of-interest and a housekeeping protein so you can normalize your data
- Image the blot on a digital imager
- Draw boxes around the bands of your protein-of-interest and your housekeeping protein and use the imager to generate a number for band intensity. Follow your imager’s instructions for subtracting background
- Calculate the ratio of your protein-of-interest to housekeeping protein to obtain relative protein abundance
With the right total protein normalization stain, imager, and chemiluminescent substrate, you can get quantitative data from chemiluminescent westerns.
3. Which of the following methods can you use to validate an antibody for quantitative western blotting:
A. Genetic method: Show that when the amount of your protein-of-interest is reduced, the signal from your antibody used in an ELISA assay is also reduced.
B. Orthogonal method: Show that measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using an orthogonal method such as mass spectrometry.
C. Independent antibody: Show that the measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using a second, already validated antibody.
Both “B” and “C” are great ways to demonstrate the specificity of your antibody, and while “A” is almost viable, the International Working Group for Antibody Validation recommends using the same technique to validate your antibody as will be used in your experiment because antibody binding is depending on experimental conditions. Thus, for antibodies used in western blotting, it’s best to validate the antibodies also using western blotting2.
4. True or False: The best way to normalize western blot data is to use a housekeeping protein?
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