Immunoblotting (Western)

 

CELL LYSATES

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.

SDS-PAGE 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.

SEMIDRY TRANSFER

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

  1. After transfering protein on to nitricellulose, put the membrane into 10 ml blocking solution and incubate at RT for 30 min.
  2. Pour off the blocking solution and add 10 ml of primary hybrization solution.
  3. Incubate for 2 hours at RT, or overnight in cold room.
  4. Wash 3 times with 1XTBST with 10 min each time.
  5. Incubate for 1 to 2 hour at RT with 10 ml of secondary hybrization solution.
  6. Wash 3 times with !XTBST with 10 min each time.
  7. rinse once with 1XPBS
  8. Briefly dry the membrane with 3 mm paper.
  9. Mix 2 ml of the two ECL reagents together.
  10. incubate the membrane with the ECL for 1 min.
  11. Briefly dry the membrane with 3 mm paper.
  12. Wrap the membrane with serawrap and put it into casetts for exposure.
    exposure time is between 30 seconds to 15 min.