Standard Operating Procedure

 

Western Blot

 

Hazards:

 

 

Protection:

Wear gloves

 

Waste: Pour waste methanol in appropriate bottle, and place in secondary containment, under the centrifuge.

 

Spill Clean-Up:

 

Procedure:

Whole Cell Lysate

  1. Spin down eppendorf tube containing the stimulated cells, carefully remove all of the supernatant.
  2. Resuspend in 50 µl Nolan 2 Lysis buffer, incubate for 30 min at 4¼C
  3. Spin and collect supernatant.
  4. Add 50 µl 2X SDS-PAGE to supernatant (w/ 2-ME).
  5. Boil in block heater at 100¼C for 10 minutes.
  6. Cool.
  7. Load 35µl into one lane (do not need to add additional SDS/2ME).
  8. Can store remaining lysate in Ð80¼C.

 

Gel

  1. Use pre-formed gels (Invitrogen, Nu-PAGE 10% Bis-Tris gel, 1.5 mm x 10 well, catalog # NP315BOX).  Remove tape and well comb.
  2. Place gel in electrophoresis chamber (wells on the inside).  Fill inner chamber with 1X MOPS running buffer, covering wells.  Add 500 µl antioxidant.  Fill outer chamber 2/3 full with 1X MOPS running buffer.

3.     Prepare ladder (Bio-Rad, Prestained SDS-PAGE Standards, broad range, catalog # 161-0318).  Add 8 µl 2X SDS-PAGE to 12 µl ladder, boil as with samples.

  1. Load 20-40 µl (36 µl optimal).  Make a written map of lanes.
  2. Run electrophoresis at 80V for 20 minutes, then 200 V until running edge almost touches bottom of gel, 60-90 minutes.

 

Transfer

  1. Prepare nylon membrane.  Using gloves, cut nylon membrane to the same size as the gel (use the template) and cut 2 sheets of 3 mm Whatman filter paper. 
  2. Soak nylon membrane in methanol for 15 seconds.
  3. Rinse nylon membrane with dd H2O.  Soak in dd H20 for 2 minutes.  Then soak in transfer buffer for 5 minutes.
  4. Prepare Xcell II blot module.  Keep everything wet!  Layer cathode plate with 2 white brillo pads, 1 sheet of Whatman, the gel, the membrane, 1 sheet of Whatman, 2 brillo pads, (for 2 gels-2 pads, 1 sheet of Whatman, gel, membrane, whatman, pad, whatman, gel, membrane, whatman, 2 pads).  Be careful to remove trapped bubbles!  Place anode core on top.  Firmly hold module together and slide into buffer chamber.  Position the tension wedge.
  5. Fill blot module with transfer buffer.  Fill outer chamber with ddH20.
  6. Run at 30 V for 45 minutes to one hour in cold room.  Can peek at membrane to see if the ladder has transferred.
  7. Remove membrane from blotting module.  Place in a weigh boat with enough blocking buffer to adequately cover the membrane.  Try to keep the side of the membrane that was in contact with the gel face-up. 
  8. Block on rocker in cold room overnight.

 

Antibodies

  1. Primary Antibody.  Transfer membrane to new weigh boat and incubate with primary antibody at appropriate dilution in 10 ml blocking buffer for one hour on rocker at RT.
  2. Wash.  In a new weigh boat rinse membrane with wash buffer, then soak in wash buffer for 5 minutes.  Repeat soaking 6 times, for a total of 30 minutes.
  3. HRP antibody.  Transfer to a new weigh boat and incubate with 10 ml blocking buffer with HRP-conjugated antibody at appropriate dilution for 45 minutes, RT, on rocker.
  4. Wash as above.
  5. Prepare chemiluminescent substrate (Pierce, SuperSignal West Pico Chemiluminescent Substrate, Catalog # 34080).  Mix equal parts of white bottle and brown bottle.  Need 10 ml for one gel.
  6. Incubate membrane in 10 ml chemiluminescent substrate for 5 min in a new weigh boat.
  7. Prepare film cassette.  Put saran wrap covered blotting paper on bottom of cassette.  Place the glowing stickers on the saran wrap.  Expose to light.  Place the nylon membrane with the side that was facing the gel face-up.  Place another piece of saran wrap on top of the membrane-smoothing out any wrinkles.
  8. Take the picture.  In the dark room, turn off the light, place a sheet of film over the membrane (smoothly, without moving the film or membrane) and cover for the appropriate length of time (15 seconds to 20 minutes).  Put film in the developing machine.

 

Stripping and Reprobing Procedure

  1. Put the membrane in a seal-a-meal bag with 10-15 ml Stripping buffer.  Incubate the membrane in the 55¼C water bath rocker for 20 minutes.
  2. Block the membrane in blocking buffer overnight.
  3. Repeat antibody steps above.

 

 

 

Reagents:

Nolan 2 Lysis Buffer (10 ml)

10 mM Tris, pH 7.5 (100 µl 1 M)

150 mM NaCl (300 µl 5M)

5 mM EDTA (100 µl 0.5M)

1% Triton-X 100 (100 µl)

1 mM Na3VO4 (50 µl 200 mM)

10 mM NaF (100 µl 1 M)

1X Protease inhibitor cocktail  (100 µl 100X stock)

H20 (9.15 ml)

 

Transfer Buffer

2 mM Tris

192 mM Glycine

10% MeOH

 

Nolan Blocking Buffer

0.1% Tween-20

1% BSA

1X PBS

 

Blocking Buffer

2.5%  non-fat dried milk (NFDM)

Wash Buffer

 

Wash Buffer                4L (in H20)

10 mM Tris                                                                                          40 ml 1 M stock (pH 7.5)

150 mM NaCl               120 ml 5M stock

0.1% Tween-20            4 ml

 

 

Stripping Buffer

62.5 mM Tris (pH 6.8) to 1 L (7.57 g)

add 20 g SDS (2%)

add 7.82 ml 0.1M 2-ME

 

Ladder

Bio-Rad SDS-PAGE Standards, Broad range #161-0318

 

Antibodies

Mouse-HRP (1:1000)               

Rabbit-HRP (1:10,000) ERK (1:1000)

Mouse pERK (1:2000)

Rabbit Oct-1 (1:200)

Hsp-90 (1:200)