I am having trouble with some final steps of my extraction. First of all I need to use a non-ionic detergent for isolation in order to maintain the protein conformation for activity assay. I won't bother you with the whole protocol, unless you need it.
The problem occurs after the 100000g centrifuge step followed by membrane homogenisation and storage at -80C. Membranes (with proteins) tend to bind to each other as they are highly hydrophobic, so after the defrosting they are not dissolved and homogenised at all, and it is a problem for quantifying the protein concentration and to run those proteins on WB or activity assay.
Membrane proteins are being isolated from primary astrocyte culture.
recommendations?
Try homogenizing the membranes with a high concentration of sucrose, such as 30%.
Why do you freeze the membranes? Is that part of the protocol or is that just to keep it until next day/until they are further processed?
well, I have to preserve them for several different protein activity and expression quantification. so first I have to determen protein concentration, then to run my samples on Western blot and also to run them for activity assay.. and everything could take several days (in my laboratory)..
@ Shruti,
thank you for your advice, but I think it is recomended for me to do sonication on ice (not in wather bath). And for trying out different detergents, I hope that I will get the chance to try them (have to order them and to wait :(..)
It seems to me that I will have to sonicate them for each aliquote that I take from deep freeze..
@ Adam,
thank you, but can you be more specific about that sucrose? how do I preserve (in sucrose?) my samples after that kind of homogenization?
I was thinking that the high viscosity and hydrogen bonding might help prevent the membranes from sticking together. I used to prepare purified plasma membrane vesicles from sonicated CHO cells by ultracentrifugation, collecting the PM vesicles from a 16%/31% sucrose interface. The membrane+sucrose solution could be stored at -80. After diluting the thawed solution into sucrose-free buffer and ultracentrifuging to pellet the membranes, I could resuspend the pellet in buffer just by using a pipet tip, resulting in a homogeneous suspension.
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