Western Blot

1. Pour the Separating Gel

Set up your gel apparatus, prepare separating gel monomer. Add TEMED just prior to pouring gel. Allow to polymerize before adding stacking gel by overlaying gently with EtOH or n-butanol.

Separating Gels, in 0.375 M Tris, pH 8.8

  7% 10% 12% 15%
distilled H2O 5.15 ml 4.15 ml 3.45 ml 2.45 ml
1.5 M Tris-HCl, pH 8.8 2.5 ml 2.5 ml 2.5 ml 2.5 ml
Acrylamide/Bis-acrylamide(30%/0.8% w/v) 2.3 ml 3.3 ml 4.0 ml 5.0 ml
25% (w/v) ammonium persulfate 0.05 ml 0.05 ml 0.05 ml 0.05 ml
TEMED 0.005 ml 0.005 ml 0.005 ml 0.005 ml
Total: 10.005 ml 10.005 ml 10.005 ml 10.005 ml

2. Pour the Stacking Gel

After the separating gel has polymerized, decant the overlay, prepare the stacking monomer, add the TEMED, and pour. Insert the comb and allow to polymerize completely before running.

Stacking Gels, 4.0% gel, 0.125 M Tris, pH 6.8

distilled H2O 3.1 ml
0.5 M Tris-HCl, pH 6.8 1.25 ml
Acrylamide/Bis-acrylamide(30%/0.8% w/v) 0.67 ml
25% (w/v) ammonium persulfate 0.025 ml
TEMED 0.005 ml
Total: 5.05 ml

3. Running the gel

1. After flash spinning the samples, load into the wells.

2. Be sure to use markers.

3. Run with constant current (35 - 37 mA with voltage set at > 300 V).

4. Usual running time is about 2.5 hr.

4. Preparation of membrane

1. Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10).

2. Wet for about 30 min in methanol on a rocker at room temp.

3. Remove methanol and add 1x Transfer buffer until ready to use.

5. Membrane transfer

1. Assemble "sandwich" for Bio-Rad's Transblot.

2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Transfer buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge

3. Transfer for 1.5 hr at 90 V with the cold pack and prechilled buffer.

4. When finished, immerse membrane in Blotto and block for 1 hr.

6. Antibodies and detection

1. Incubate with primary antibody diluted in Blotto for 2 hr at room temp (or overnight at 4°C).

2. Wash 3 x 5 min with PBS 1 x 5min.

3. Wash 5 min. with PBS containing 0.05% Tween 20 in PBS(PBST).

4. Wash 5 min with PBS 1 x 5min.

5. Incubate with secondary antibody diluted in Blotto buffer for 1 hr at room temp.

6. Wash 3 x 5 min with PBS 1 x 5min.

7. Wash 5 min. with PBS containing 0.05% Tween 20 in PBS(PBST).

8. Wash 5 min with PBS 1 x 5min.

7.Detect with Amersham ECL kit

1. Materials needed: Two pieces of saran-wrap, detection reagent 1 and 2, film cassette, scissors, tweezers and film.

2. Mix 2ml of 1 and 2ml of 2 in a 15ml tube (BE SURE TO CHANGE PIPETS IN BETWEEN REAGENTS).

3. Use tweezers to move membrane on to a piece of saranwrap and drip the reagent mix all over the membrane. Let stand for 1min.

4. Drip off excess mix, and transfer the membrane to a clean piece of saranwrap upside down. Wrap the membrane up in the saranwrap and place it into the film cassette right-side up.

5. Bring the cassette, film and scissors in a darkroom with a developer. When in the darkroom, cut a piece of film to fit the cassette.

6. Expose the film for 1min, bend the top right hand corner down and back again to distinguish the correct orientation.

7. Slide the film through the developer to develop.

8. Stripping the blot

1. Strip antibodies by incubating blot for 30-90 minutes at 70°C in Stripping Buffer

2. Wash 2 times, 10 minutes each wash, in PBST Buffer.

3. Block for 2.5 hours in Blotto.

Buffers for Westerns

Resolving gel buffer: 100 ml
     0.4 g SDS (add last)
     18.2 g Trizma base (= 1.5M)
     Adjust pH to 8.8 with concentrated HCl

Stacking gel buffer: 100 ml
     0.4 g SDS (add last)
     6.05 g Trizma base (= 0.5 M)
     Adjust pH to 6.8

10x Running buffer: 1 L
     30.3 g Trizma base (= 0.25 M)
     144 g Glycine (= 1.92 M)
     10 g SDS (= 1%)--add last
     Do not adjust the pH!!

10x Transfer buffer: 1 L
     30.3 g Trizma base (= 0.25 M)
     144 g Glycine (= 1.92 M)
     pH should be 8.3; do not adjust

To make 2 L of 1x Transfer buffer:
     400 ml Methanol
     200 ml 10x Transfer buffer
     1400 ml water

Blotto: 0.5 L
     2.5% Nonfat dry milk powder
     Make up in PBS.
     Then add 0.05% Tween 20.
     Keep at 4°C to prevent bacterial contamination.

Stripping buffer: 0.5 L
     62.5 mM Tris-HCl pH 6.8
     2% (w/v) SDS
     100 mM b-mercaptoethanol

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