Reagents:
1. 2x RIPA buffer*
Reagent [final] [stock] volume, stock
Tris Cl pH 7.4 50 mM 1.0 M 25 ml
Triton X-100 1% 100% 5 ml
sodium deoxycholate (DOC) 1% solid 5 g
SDS 0.1% 10% 5 ml
EDTA, pH 7.0 1 mM 500 mM 1 ml
NaCl 150 mM 5.0 M 5 ml
dH20 459 ml
500 ml
*add to 2x stock just before use:
PMSF (Sigma) 1 mM 500 mM 1:500
in DMSO
aprotonin (Sigma) 1% 100% 1:100
solution from Sigma
pepstatin (Sigma) 10 µg/ml 10 mg/ml 1:1000
in EtOH
Na3VO4 1 mM 50 mM 1:50
NaF 50 mM 500 mM 1:10
4x gel loading buffer
Reagent [final (1x)/(4x)] [stock] ml stock
Tris-Cl pH 6.8 60 mM/240 mM 1M 6.0 ml
2-mercaptoethanol 5.00%/20% 14.7 M 5 ml
SDS 2.0%/8.0% dry 2.0 g
glycerol 10%/40% 100% 10 ml
bromphenol blue .05%/0.2% dry 12.5 mg
dH20 as required
25 ml
Procedure
1. Lysis of cells by directly boiling: Resuspend cells in 1XSDS loading
buffer at about 300 ul per 107 cells and boil them for 5 minutes. Spin in the
microfuge before loading the gel.
2. Lysis of cells by RIPA buffer: Resuspend cells in 1X RIPA buffer at about 300 ul per 107 cells and incubate on ice for 30 minutes with mixing by pipetting up and down for several times. Spin at 10000g for twenty minutes at 4 C in the microfuge and transfer the supernatants to a fresh tube, with care not to dislodge the viscous pellet formed after centrifugation. Add 100 ul of 4X Loading buffer and boil the lysates for 5 minutes before loading the gel.
Separating: Per 10 ml (approx. 7.5 mls/1.5 mm gel, 5 ml/1 mm gel)
% 7.5 9 10 11 12 13
30% Protogel 2.27 mls 3 ml 3.33 mls 3.67 mls 4 mls 4.33 ml
10 X Sep 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Water 6.58 6 ml 5.52 5.18 4.85 4.52
10% SDS 100 µl 100 µl 100 µl 100 µl 100 µl 100
µl
10% APS 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl
TEMED 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
Recommended acrylamide gel percentages: 7.5% for 40-200 kD proteins; 9% for 30-150 kD; 10% for 21-100 kD; 11% for 15 to 60 kD; 12% for 10-40 kD; 13% for 6-30 kD.
Stacking: Per 10 ml (approx. 2.5 mls/gel)
% 4 5
30% Protogel 1.33 ml
10 X Stack 1 ml
Water 7.5 ml
10% SDS 100 µl
10% APS 100 µl
TEMED 10 µl
Load 50 ug of total proteins from cell lysate or 20-100 ng purified protein
into a 1 x 3 mm gel slot. Larger volumes are not much a problem, with up to
15 µl possible without smearing when run at voltages of 100-125 V constant
voltage or 25 mA constant current in minigel apparatus. Prestained markers can
be used conveniently to estimate transfer in subsequent steps. Run the gel till
bromphenol blue just off end of gel.
Reagents:
#1. transfer buffer
Reagent [final] [stock] volume, stock
MeOH 15% 100% 15 ml
10 x running buffer, w/o SDS 1x 10 x 10 ml
dH2O 75 ml 100 ml
#2. Running buffer without SDS, 10x stock solution
Tris base, 30 grams/liter
glycine, 144 grams/liter
Procedure
1)Soak gel in transfer burrer for 5 minutes; wet nitrocellulose membrane in water first, then transfer buffer while gel is soaking.
2) Set up transfer apparatus:
3) Run 15 V x 30 minutes.
WESTERN HYBRIDIZATION
Reagents
Procedure