protein separation - (Nov/27/2006 )

Hi,

Can anyone tell me how to separate transmembrane (or membrane-bound) proteins and cytoplasmic proteins when isolating proteins from cells? Any advice would be really appreciated!

Hi

just curious to know if you are working with a known protein or an unknown protein and why you need to separate membrane bound proteins and cytoplasmic proteins

i might be wrong but as far as i know membrane proteins are lipid soluble and cytosolic protiens are water soluble so......so first treat your cells without any detergent and then add detergent to the pellet and disolve it (this is membrane bound proteins). please someone correct me if im wrong.

use differential centrifugation; after lysis or for largher amounts, pottering,
run first at 80xg for debris, then the supernatant at 100.000xg to separate cytosolic from microsomal proteins;

if you have larger amounts you can utilize also a combination of differential centrifugation comprising sucrose gradients

Thanks to all who have replied. I want to look at a certain transmembrane protein. I have so far done protein isolation by 1)lysing the cells with RIPA buffer (with protease inhibitor), 2) centrifuging at 4C for 10' at 8000 rpm, and 3) collecting the supernatant (and discarding the pellet). With the proteins isolated in this way, I did western blot and did not detect the protein of interest. My PI suggested me to look at the transmembrane proteins. This is why I ask here. I would really appreciate it if anyone can give me a detailed protocol, e.g. how much SDS (and what percentage) should I add to the pellet, how long should I wait, vortex? determine the concentration? etc...

Thank you very much!!!

[quote name='myco_atgc' date='Nov 27 2006, 07:35 PM' post='78672']
Hi

just curious to know if you are working with a known protein or an unknown protein and why you need to separate membrane bound proteins and cytoplasmic proteins

Hi

you have mentioned that you discard the pellet after lysing the cells. Why dont you take the sup out resuspend the pellet in lysis solution and run it parallely along with the supernatant. Also check if your antibodies working.

good luck


[quote name='wjchxl' date='Nov 28 2006, 01:43 PM' post='78840']
Thanks to all who have replied. I want to look at a certain transmembrane protein. I have so far done protein isolation by 1)lysing the cells with RIPA buffer (with protease inhibitor), 2) centrifuging at 4C for 10' at 8000 rpm, and 3) collecting the supernatant (and discarding the pellet). With the proteins isolated in this way, I did western blot and did not detect the protein of interest. My PI suggested me to look at the transmembrane proteins. This is why I ask here. I would really appreciate it if anyone can give me a detailed protocol, e.g. how much SDS (and what percentage) should I add to the pellet, how long should I wait, vortex? determine the concentration? etc...

Thank you very much!!!

[quote name='myco_atgc' date='Nov 27 2006, 07:35 PM' post='78672']
Hi

just curious to know if you are working with a known protein or an unknown protein and why you need to separate membrane bound proteins and cytoplasmic proteins
[/quote]

read the first page of this pdf
http://www.abcam.com/assets/pdf/protocols/WB-beginner.pdf

thanx for the link