Am 03.03.2010, 07:38 Uhr, schrieb ketut gunarta <[email protected]>:
Hello all,
I have to isolate G-PCR (G-protein coupled receptor) from ovarian cancer
cell line. I am looking for a protocol (buffer,etc) that not use
extraction/isolation kit (I could mix the buffer by my self) and not
change its native structure. There is a problem that my ultracentrifuge
only can spin up to 15,000 g. I hope anybody could help me. Thank you
very much.
15,000 g is not an ultracentrifuge, but a clinical one. This is
insufficient for your task. With a proper ultracentrifuge you could do
something like this:
Harvest your cells with EDTA only (no trypsin, you don't want to chew your
receptor). Spin down at 300 g for 10 min. Resuspend in a minimum volume of
supernatant and transfer to a tube for an ultracentrifuge rotor. Add
dropwise ice-cold swelling buffer (10 mM Tris or HEPES pH 7.3, protease
inhibitors, ßME) under constant swirling, until tube is full. Incubate on
ice for 30 min. Seal and run 150,000 g 30 min in a fixed angle rotor. The
pellet contains "cell ghosts". Cytosol, lysosomes, mitos etc. are in the
supernatant. That way you get rid of most of the proteolytic activity
early.
The ghosts are homogenised in a Potter in 250 mM Sucrose in swelling
buffer. Spin for 100 g 10 min to remove nuclei, unhomogenised cells etc
and load the supernatant onto a 35% sucrose cushion and spin in a swinging
bucket rotor at 50,000 g for 30 min. The cell membranes will be at the
interface to the cushion. Isolate, dilute 3-fold with swelling buffer and
spin down 85,000 g 45 min. This crude preparation of cell membranes can
then be used as starting material for solubilisation with detergent and
protein purification. You can also snap-freeze the membrane suspension in
swelling buffer (liquid N2) and store at -80 °C.
For solubilisation you have to test what works best, use published papers
in your field as guideline. You can calculate the detergent concentration
needed for solubilisation the following way:
You need about 10 detergent micelles per protein molecule. For the average
molecular weight of membrane proteins you can assume 100 kDa, thus a 1
mg/ml protein preparation is about
10 µM, and you need a micelle concentration of 100 µM to solubilise them.
One detergent micelle can solubilise about 10 lipid molecules. Assume that
a preparation
containing 1 mg/ml membrane proteins will be about 1 mg/ml in lipid with
an average
molecular mass of 750 Da. Thus for a 1 mg/ml protein preparation you need
a micelle concentration of 130 µM to solubilise the lipid.
To solubilise both lipid and protein from a 1 mg/ml membrane protein you
need a micelle concentration of 230 µM. To get a micelle concentration of
230 µM, you need
{\bar m} * 230 µM + cmc detergent, with {\bar m} the aggregation number
and cmc the critical micellar concentration. These data you can look up
for example in a handbook published by Calbiochem.
Do solubilisation experiments in, say, 100 µl, using 1/4, 1/2, 1, 2, and 4
times the calculated detergent concentration. Keep your sample on ice for
30 min, then spin at 100,000 g for 1 h in a desktop ultracentrifuge with
rotor for 200 µl tubes. Measure recovery of protein and activity in the
supernatants. Do this for different detergents to find those that work
with your protein. You may repeat the activity measurements after a
weekend, to account for the time it takes to purify your protein.
Once you have your protein solubilised, you can purify by conventional
methods, but make sure that you include the detergent at the cmc in all
buffers. The cmc depends on buffer composition, to detemine it use the
following method:
@ARTICLE{Cha-84,
AUTHOR= {A. Chattopadhyay and E. London},
TITLE= {Fluorimetric determination of critical micelle
concentration avoiding interference from detergent charge},
JOURNAL={Anal. Biochem.},
YEAR= {1984},
VOLUME= {139},
PAGES= {408-412}
}
One little hint: Positively charged detergents like CTAB are cheap, very
effective in protein solubilisation but gentle to protein activity. CTAB
can also be used for electrophoresis and blotting instead of SDS:
@article{Bux-03,
AUTHOR= {E. Buxbaum},
TITLE= {Cationic Electrophoresis and Electrotransfer of Membrane
Glycoproteins},
YEAR= {2003},
JOURNAL= {Anal. Biochem.},
PAGES= {70-76},
VOLUME= {314},
DOI= {10.1016/S0003-2697(02)00639-5}
}
This is also the reference for the isolation of plasma membranes by the
above method.
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