How to prepare plasma-membrane fraction? - (Jan/29/2009 )

Hi all,
I would a few questions regarding proteins localized in plasma-membrane (of mammalian cells) and preparation of fraction containing plasma-membrane. A protein of my interest (60kDa) should be typically localized in plasma membrane. I have two different antibodies for detection of the protein.
I do homogenization of scraped cells by Potter-Elvehjem homogenizator (glass tube with teflon pestle), then centrifugation at 1000, 14000, 110 000g in PBS containing 0.25M sucrose. The first antibody detects (my) protein mainly in supernatant after 110 000g centrifugation (90% of the protein is in supernatant and 10% in pellet) and the Mw of the protein is 55kDa. The second antibody (against the same protein) detects a protein (60kDa) mainly in pellet after 14000g centrifugation.
I am confused from these results. May be, you can give me some advices or suggestions to my questions.
If the protein is in supernatant after 110 000g centrifugation, is it likely not (integral) plasma-membrane protein?
If a protein is in pellet after 14000g centrifugation, is it like not plasma-membrane protein?
Is the working-protocol that I use for preparation of fraction containing crude plasma-membrane correct?

Unfortunately, I do not have well functional siRNA to show what and where my protein is.

Thanks.
vic

Do you know if this protein can be for example cleaved or further processed and then excreted to the cytoplasm ? This 5KDa difference might well be what has been kept stacked in the membrane

I also do cellular fractionations but I extract mitochondria and as far as I'm concerned plasma membrane is only separatable at very high speed.

my adviser told me not to use any type of homogenizer (in my case Dounce homogenizer) because homogenizers are not suitable for localization studies, maybe you need to avoid homogenization too!

I use digitonin lysis buffer and it works for my case. I do not put cells under so much homogenization pressure.

have a look at this paper, it might help:
Samali et al. 1999. The EMBO Journal Vol.18 No.8 pp.2040–2048

Thanks for your suggestions.
As I said, my protein (according literature) should be localized in plasma membrane, but I detect this protein in supernatant after 110 000g centrifugation, which is mainly cytosol (?). But interestingly, when I did centrifugation at 200 000g the protein was in the pellet (cells where lysed in PBS by Potter-Elvehjem homogenizator).
What is the conventional force (speed) to pellet plasma membrane - is it 100 000g?
Why, according some protocols, it is sometimes 150 000 or 200 000g?

vic

just read the paper I gave you

Curtis, thanks for the paper.
I have looked at technical parts of the paper and some figures. There are some interesting information and there is nice centrifugal separation of nuclei, mitochondria, cytosol, microsomes. But my protein should be in plasma membrane, so I am particularly interested in plasma membrane separation. I suppose that the plasma membrane is mainly in microsomal fraction (100 000g pellet). But in some papers, I have found, that the force of 150 000g or 200 000g (instead of 100 000g) was used to pellet plasma membrane. In this regard, I am also interested in practical experience how someone else is preparing fraction containing plasma membrane.

vic

um....I always thought the microsomal fraction is the last fraction because in this paper the membrane is broken down by digitonin into single proteins (not entirely but still it is not an intact membrane anymore)....please correct me if I'm wrong.

where is rkay447?

try this paper too, but this one is with homogenizer.

http://www3.interscience.wiley.com/journal/69501035/abstract