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First described in 1979, the technique of western blotting has since become one of the most commonly used analytical methods in life science research. Just last week, we received a few questions from confused researchers about weird band sizes in their western blot results:
Let's try to clear up some confusion surrounding western blot data analysis. As you may already know, western blotting is a technique that separates proteins based on size using gel electrophoresis and buffers loaded with sodium dodecyl sulfate (SDS). When analyzing the western blot results, the expected band size is predicted based on the size of the protein. If you know the protein’s primary amino acid sequence, then you can calculate what molecular weight (MW) the protein is predicted to be.
Here is the key information you need to remember: The average molecular weight of an amino acid is 110 Daltons (Da). For example, if your protein sequence is 170 amino acids long, we predict the MW of the protein will be 170 x 110 Da = ~18,700 Daltons (or 18.7 kDa).
In general, the smaller the protein, the faster it migrates through the gel. However, migration is also affected by a few other factors. As a result, the actual band size observed in your results may differ from what was predicted by math. These other factors include, but are not limited to:
Different types of post-translational modifications
Aquaporin-1’s expected band is 28kDa. However, after AQP1 glycosylation, another band is observed around 50kDa.
Now that you understand why you might observe different band sizes than what is expected, you may be wondering, “How can I find out exactly what is going on with my specific antibody?”
When you encounter this situation, the easiest first step would be to contact the antibody manufacturers and ask why they detect different band sizes. At Bosterbio, over 97% of customer support requests are satisfied within 24 hours, so don’t hesitate to contact us by phone, email, or the live chat feature on our homepage. Next, you could check online for some publications in the field to figure out:
We hope this information will help you gain more insights about your western blot results. If you have any further questions, don’t hesitate to contact [email protected] for more information.
Learn more about Western Blot Principle, Protocol, Optimization, and Troubleshooting