I am working with histamine receptor 1, a G protein couple receptor (GPCR), and when i prep my lysate for SDS page gel run I heat them up at 95C/3 min. I am getting protein aggregation in my western bands. I tried 65C/10 min and still the pattern isn't changed. Has anyone worked with H1R and used any strategy to get around this problem.
I am not sure if its the glycosylated H1R that is causing this multimer pattern of GPCR
Donot boil or heat the sample; this is what my colleagues who work with GPCRs suggest and do. They are not sure why this works. It likely prevents aggregation. Try it out to see if this improves your western blots.
Common problem for transmembrane protein actually (not just HRH1), check this out:
http://www.ncbi.nlm.nih.gov/pubmed/17623834
http://www.scientistsolutions.com/t1140-western+blotting.html
https://www.researchgate.net/post/Plasma_membrane_proteins_and_western_blot2
Hope that help,
Radif
Donot boil or heat the sample; this is what my colleagues who work with GPCRs suggest and do. They are not sure why this works. It likely prevents aggregation. Try it out to see if this improves your western blots.
It could be a glycosylation issue. Incomplete glycosylation could lead to aggregation, which is common with membrane proteins. You might need to screen for a better expression host. However, it is best to start with not boiling the lysate at all and see if that makes a difference.
I agree with Ankita's ideas and suggestions. You do not mention the expression system used but I would suggest looking at reducing levels of expression in your current system or any others that you consider. Issues with glycosylation and other aggregation issues can be caused or exaggerated by very high expression overloading certain pathways. Sometimes less is more. If you are using a transient system consider harvesting earlier or lowering levels of stimulus. If it is a stable system consider lower expressing clones. A perfused system may help to reduce stress caused by any overloading of particular pathways. Partially purifying the membrane fraction is also likely to help. As suggested by others, I would definitely look at a range of temperatures for treating lysates of whole cells or fractions. It's very difficult to predict which would be best. Low temperatures are often optimal, but not always!
i am testing the room temperature incubation and will let you know the findings. thanks for the comments.
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