Hi Zhongliang!
I think there are pretty good protocol papers out there...
One of my favorite is by Drew et al. 2008 (http://www.nature.com/nprot/journal/v3/n5/full/nprot.2008.44.html).
Good luck!
Yoni
The paper above largely focuses on purification from yeast. What system are you try to isolate your membrane protein from?
DR. Brandon
FOCUSE ON CANCER CELL. WOULD YOU HAVE SOME POSITIVE ADVICES?
I assume by cancer cell, you mean mammalian cells.
This is actually often a straight forward protocol if you protein is well behaved, and usually construct optimization is the key.
You should harvest the mammalian cells in a isotonic buffer.
1) Collect the cell pellet.
2) Lyse the cells in a hypotonic buffer (I usually use 20mM HEPES pH7.4/2mM MgCl2 with 1:100000 benzonase).
3) Spin the lysate at ~ 18000 rpm in ss34 rotor or similar for 15minutes.
4) Recover the pellet (you can discard the supernatant). Take a sample of the supernatant to make sure your protein is in the membrane/insoluble fraction.
5) Solubilize the pellet in the appropriate detergent - usually 1% DDM or 0.5-1%L-MNG to start (dissolved in 20mM HEPES, 150mM NaCl pH7.4). The volume should be equal to 10X the cell paste weight.
Use a dounce homogenizer to really break up the pellet - 10x pestle A and 10x pestle B. (Save 50ml of solubilization buffer for future steps)
6) Solubilize for ~2 hours - try to be consistent - do not leave overnight.
7) Spin @ 20000rpm for 20 min in ss34 rotor or similar.
8) Filter the supernatant over a glass fibre filter to get rid of any particulate or "goop" before binding to appropriate resin.
If using NiNTA - batch bind for 1-2 hours at 4 deg. If using Flag or ProtC resin, bind by gravity.
9) Wash with 0.1X solubilization buffer ~ 50mL.
10) Elute in 1mL fraction using 0.1X solubilization buffer with appropriate elution conditions.
Use a nanodrop or protein dye - to test each fraction from the presence of protein.
Assess quality by coomassie gel or western blot.
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases
- Immunization
- Vaccination