protein purification - (Jan/28/2009 )
is it possible to purify cytosolic, nuclear and membrane proteins seperately from the same tissue?
I have tissue samples that are very precious, don't have much, and need to do westerns on all three compartments.
I found a ready to use kit, but you need a lot of starting material for that.
I suppose I'm a bit confused as to what you want to do but if all you want is westerns you can make lysate from the whole tissue and do the westerns on whole cell extract. You don't neccessarily need to purify the proteins before doing the westerns. If you do wish to isolate each protein I would recommend IPs. You could try to make extract from the tissue and do each individual IP in tandem from the same lysate. Never heard of this being done but I don't see why it can't be done as long as you have good antibodies.
I would suggest fractionation by centrifugation / ultracentrifugation. After opening cells in mild detergent (like NP-40) nucleous form pellet at low RFC (around 3000 to 5000 g). If you do ultracentrifugation on your fist supernatant you will be able to separate membranes from cytosolic fraction.
Of course, this is the theory, and practically you will never get complete separation, but you will greatly saturate each fraction with corresponding cell compartments.
Let me know if you want more information on it.
Sorry if I didn't express myself clearly.
I want to do westerns, but on the seperate fractions of the lysate. So I need some kind of ultracentrifugation protocol.
And I want one fraction with the nuclei, one with cell membranes and one with cytosolic proteins.
General guidelines are next:
1. After tissue homogenization you should clarify the lysate from unbroken cells and stromal components.
That should be very low speed centrifugation because nucleus can sediment at very low "g". I would start from 100 g for 5 min.
2. Next you will need to pellet down nucleus. There is a plenty of protocols for nucleus isolation (like this for example: http://www.lamondlab.com/f7nucleolarprotocol.htm) I would make 0.3M sucrose cushion and centrifuged for 1500 g for 10 min. In pellet you will have fraction saturated on nucleus and part under sucrose cushion will contain cytosolic proteins and membranes.
3. Last I would pellet down membranes by centrifugation of supernatant form step 2 (part under cushion) at 100 000 g for 2 hr (you need ultracentrifuge for that). Pellet will be saturated on membrane proteins, where as supernatant will contain cytosolic proteins.
Keep in mind, that is not purification, but just a saturation. You need control each fraction for cross-contamination with well established nucleus, cytosolic and membrane protein markers. If you want a purify fraction, you should do fractionation by gradient density centrifugation.
PS You know, it is better ask such a question on Cell Biology forums
I have a question on a similar lines.
I am expressing a protein in Sf9 cells. My protein is supposed to be in ER fraction.
What I basically do is:
1) Resuspend the cells in Phosophate buffer with protease inhibitor.
2) Sonicate the cells at 30% amp, 10 x 5 secs, with interval of 15 secs.
3) Spin down at 3000 rpm for 5 min, to get rid of debris.
4) Take supernatent and spin at 70.000 rpm for 45 min (thats d max. speed my ultracentrifuge can afford)
5) Finally resuspend in hepes-naoh ph 7.0
6) Pass it through 22 guage needle at least 10 times.
Problem here is I cannot resuspend it entirely in Hepes-NaOH.
How can I increase the solubility of the membrane fraction?
Any suggestions for modifying the protocol also would be appreciated.