protocol for membrane receptor isolation

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Tue Jun 24 14:45:56 EST 2008


Am 23.06.2008, 10:03 Uhr, schrieb Behdani <behdani from pasteur.ac.ir>:

> Hello All
> I have to isolate cytoplasmic membrane receptor from 293 cell line  
> (Human Kidney) to immunize rabbit for polyclonal antibody production. I  
> am looking for a protocol (lysis buffer and other steps) which contains  
> reagents which save native form of membrane proteins. Could somebody

The following recipe works with cells from 10 20x20 cm2 plates or a "cell  
factory" of similar area. The idea is to hypotonically lyse the cells and  
spin down the "ghosts", without all the proteases that are in the cytosol.  
The ghosts are homogenized and the plasma membranes purified by a crude  
sucrose step gradient centrifugation.

- First of all, do not use trypsin/EDTA for harvesting, 5 mM EDTA in PBS  
is quite capable of detaching cells and does not digest your proteins.  
Spin down your cells.

- Transfer cells in a minimal volume to a tube for 50.2Ti rotor and add  
dropwise ice-cold swelling buffer (10 mM Tris, 5 mM 6-aminocaproic acid, 1  
mM beta-mercaptoethanol, 100 µM PMSF (added immediately before use from 1  
M stock in i-propanol), 100 µM EDTA, 100 µM benzamidine, 1 µM leupeptin, 1  
µM pepstatin and Hepes to pH 7.3) under constant swirling until the tube  
is filled (39 ml final volume). Keep on ice for 30 min, then spin at  
40,000 rpm (145,000 g), 4 C for 30 min.

- Discard the supernatant and take up the cell ghosts in a few ml of  
homogenisation buffer (10 mM Tris-HCl pH 7.4, 250 mM sucrose with protease  
inhibitors as above). Homogenise in a Potter- or Dounce-homogenizer and do  
a crap-spin at 100 g for 10 min.

- Layer the supernatant onto a cushion of 35% sucrose in 10 mM Tris-HCl pH  
7.4, 0.1 mM EDTA and spin for 35 min at 50,000 g in a Beckman SW25 rotor.

- Collect the band between sample and cushion by aspiration, dilut 3 fold  
with homogenisation buffer and spin down in a Ti70 at 85,000 g for 45 min.

- Resuspended the pellet in homogenisation buffer (-PMSF) at 5 mg/ml  
protein and snap freeze aliquots in liquid nitrogen. They keep well at -80  
C.

The protease inhibitor mix above (EDTA, 6-aminocaproic acid, PMSF,  
benzamidine, leupeptin and pepstatin) worked well for me, but you may have  
to adapt it to your particular protein. Commercial mixes often do not  
contain 6-aminocaproic acid, which I found essential.


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