My immunoblotting failed to detect a protein of interest and I'm using the M-PER™ Mammalian Protein Extraction Reagent, which could efficiently extract both cytoplasmic and nuclear proteins whereas perform poorly in extracting membrane-bound protein. I was wondering if simply boiling the cell pellets in SDS loading buffer could make any difference. Thanks
People sometimes find that it is better not to boil membrane proteins after adding SDS-PAGE sample buffer.
try to use ultra sound after you isolated nuclear and cytoplasmic samples. Just shortly on ice (or icy ultra sonic bath) and repeat it for at least three times. That should work. Worked at least with my Protein of interest
Yes you can start by resuspending a total cell pellet in SDS-Sample buffer, and you can boil for 5 minutes, but you can also incubate at room temperature for 5 minutes, and some conditions inbetween. Adam is right that for some membrane proteins, boiling can lead to fragmentation. First keep it simply and focus on detection, ideal is if you have positive and negative controls for your western, but that's not always possible. A pisitive control would be the purified protein that was used to raise the antibody. A negative control is any cell extract that doesn't have your protein (E.coli with empy vector, or mammalian knockout cell line). You need to know that your western blot is working, then you can interpret the extraction from the test objects.
In general, when you work with a new membrane protein, you have to try different methods and keep side fractions. So if you sonicate with an extraction buffer and spin, don't just analyse the supernatant, but also test the pellet (extract with SDS-sample buffer) and try different extraction buffers.
Yes, it is possible. However, some membrane proteins such as GPCRs aggregate when boiled the sample. So try initially boiling the cell pellet resuspended in 1X SDS-PAGE buffer and if it fails, then incubate the sample at room temperature for 1 hour or at higher temperature (50-60C) for 15-20min. We had to use incubation at room temperature rather than boiling the sample to see GLP-1R membrane protein expression by immunoblotting (http://www.nature.com/srep/2014/141215/srep07410/full/srep07410.html).
I also had problem with some membrane (ER) protein. The best conditions for me were to boil samples for 5 minutes in increased (4%) SDS concentration.
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