I need some advise in purifying a membrane protein which is expressed in mammalian system HEK293.
Provided that you are sure that the protein is in the membrane, you should obtain that fraction by centrifugation and then you should try solubilization using a detergent. The use of which one depends of the properties of the protein. SDS is most effcient, but the protein would be denatured. Alternatively, you can use a non-ionic detergent, such as Igepal, Tween.....
Provided that you are sure that the protein is in the membrane, you should obtain that fraction by centrifugation and then you should try solubilization using a detergent. The use of which one depends of the properties of the protein. SDS is most effcient, but the protein would be denatured. Alternatively, you can use a non-ionic detergent, such as Igepal, Tween.....
We used cell disruption in 0.25M sucrose, HEPES buffer, followed by sucrose gradients. You can look up how to isolate CYP450 enzymes from liver. But basically, one prepares a sucrose gradient from 38%-43% sucrose w/w and then layers a suspension of the particulate matter from disruption onto the sucrose gradient and then spins at 100,000 xg. So one would first disrupt cells/ tissue in the 0.25M sucrose buffer with protease inhibitors, spin at 100,000xg and then resuspend the pellet in the 0.25M sucrose buffer, and then layer on top of the gradient. If you assay the different fractions, and find your activity, you can then try different detergents to solubilize your protein.
This Nature Protocols paper has some details near the end that you can follow.
http://www.nature.com/nprot/journal/v7/n3/full/nprot.2011.453.html
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