24 Protein Techniques
373
acids should never be used because oftheir low specific activity (300 mCi/
mmol) and high cost.
Celllysis
Cells can be lysed with a variety of detergents. SDS (0.5-l.Oo/o) will disrupt
the nuclear membrane and should be followed by either enzyme treatment
or shearing ( sonication) to disrupt DN A prior to the addition of the primary
antibody.
Sampie immunoprecipitation protocol
The following is a sample protocol for a membrane protein. No single protocol will be appropriate for all antigens.
I. Prepare Selectamine media (Gibco-BRL), that is deficient in the amino
acid to be labeled, according to the instructions provided by the manufacturer.
2. Add dialyzed fetal calf serum to a final concentration of 10%.
3. W ash the cells in this medium.
4. Add the isotope to the smallest possible volume of media ( 10 mls for a T175 flask), filter, and place in the flask overnight.
1. Rinse the cells in 10 ml ofK-Hepes solution (200 mM mannitol, 41 mM
K+, 80 mM N-2-hydroxyethylpiperazine-N' -2-ethenesulfonic acid, (pH
7.5)) containing the protease inhibitors Ieupeptin (0.5 f.lg/ml), pepstatin
(0.7 f.lg/ml), phenylmethylsulfonylfluoride (PMSF) (40 f.lg/ml), and
EDTA (lmM) (solution A) and harvest by scraping with a disposable
plastic policeman.
2. Rinse the flask with an additional 10 ml of solution A. Centrifuge at
2,300xg for 10 min. Discard the supernatant and freeze the pellet in
an ethanol-dry ice bath.
3. Resuspend the pellet in 10 ml of solution A and homogenize 40 strokes in
a Potter-Elvehjem homogenizer.
4. Centrifuge the homogenate at 2,300 x g for 10 min.
5. Collect the supernatant and centrifuge at 23,600 x g for 1 hr. Resuspend
the pellet in 100 f.ll of solution A (protein concentration approximately
15-25 mglml).
Metabolie labeling
with amino acids
Preparation of
microsomal
membranes
373
acids should never be used because oftheir low specific activity (300 mCi/
mmol) and high cost.
Celllysis
Cells can be lysed with a variety of detergents. SDS (0.5-l.Oo/o) will disrupt
the nuclear membrane and should be followed by either enzyme treatment
or shearing ( sonication) to disrupt DN A prior to the addition of the primary
antibody.
Sampie immunoprecipitation protocol
The following is a sample protocol for a membrane protein. No single protocol will be appropriate for all antigens.
I. Prepare Selectamine media (Gibco-BRL), that is deficient in the amino
acid to be labeled, according to the instructions provided by the manufacturer.
2. Add dialyzed fetal calf serum to a final concentration of 10%.
3. W ash the cells in this medium.
4. Add the isotope to the smallest possible volume of media ( 10 mls for a T175 flask), filter, and place in the flask overnight.
1. Rinse the cells in 10 ml ofK-Hepes solution (200 mM mannitol, 41 mM
K+, 80 mM N-2-hydroxyethylpiperazine-N' -2-ethenesulfonic acid, (pH
7.5)) containing the protease inhibitors Ieupeptin (0.5 f.lg/ml), pepstatin
(0.7 f.lg/ml), phenylmethylsulfonylfluoride (PMSF) (40 f.lg/ml), and
EDTA (lmM) (solution A) and harvest by scraping with a disposable
plastic policeman.
2. Rinse the flask with an additional 10 ml of solution A. Centrifuge at
2,300xg for 10 min. Discard the supernatant and freeze the pellet in
an ethanol-dry ice bath.
3. Resuspend the pellet in 10 ml of solution A and homogenize 40 strokes in
a Potter-Elvehjem homogenizer.
4. Centrifuge the homogenate at 2,300 x g for 10 min.
5. Collect the supernatant and centrifuge at 23,600 x g for 1 hr. Resuspend
the pellet in 100 f.ll of solution A (protein concentration approximately
15-25 mglml).
Metabolie labeling
with amino acids
Preparation of
microsomal
membranes