Western Blotting

1. Optional: "Renature" gel- this is thought to permit some refolding of proteins and may be important in finding epitope recognition of monoclonal antibodies. However its major advantage is probably that it removes the SDS from the gel which impedes transfer.

Soak for 30 min- 2 hrs in: 10 mM Tris pH 6.8

0.1 M DTT

4 M Urea

50 mM NaCl

2. Transfer 2-3 hrs- O/N at 350mA

3. Block in TBS containing 0.05- 0.1% Tween for 15 mins to O/N OR

+ 5% non-fat dryed milk for ECL at RT for 2hrs

4. Stain with Ponceau S for 5 mins

5. Dilute antibodies in blocking buffer.

1/500 for 125I blots for 2hrs RT

1/-5,000 for flag & myc and 1/3000 for 5172 for ECL O/N at 4C

6. 3 X 10 min washes in TBS-Tween.

7. Incubate in Rabbit anti-mouse 1-2 hr at RT (dilute 1/500)

8. 3 X 10 min washes in TBS-Tween.

9. Secondary antibody 1-2 hr at RT

(Use NEX-L- low specific activity 125I-protein A -diluted 2 x 105cpm/ml

(1uCi = 106 cpm)

dilute Goat anti mouse HRP-ab 1/4,000 for ECL

Add 1M NaCl and 0.5% Triton X-100 to the middle wash for 30 mins to 1 hr if background is a problem.

ECL

Incubate 1min @ RT in mix (1:1) of the two ECL solutions

Dry out the excess liquid on Whatman paper

Wrap the membrane in Saran wrap

10X Tranfer Buffer

30.27g/L Tris 8.8 -25mM

144.1g/L Glycine -192 mM

ECL reagent: homegrown version

Advantages: almost no cost; stable/reproducible, stored in frozen aliquots

To 10 mls of 100 mM Tris pH 8.5 (RT), add

1. 50 ls of luminol (warm to redissolve)

2. 22 ls of coumaric acid (warm to redissolve)

3. 3 ls of H₂O₂ (fresh < 6 months)

Pour onto blot for 1 minute and process as normal (10 mls enough for 100 cm²)

Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266 mgs in 6 mls DMSO; store frozen in 60 ls aliquots.

Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in 2.5 mls DMSO; store frozen in 25 l aliquots.

Stripping blots:

Most antibodies should be removed by heat and SDS:

100 mls

-ME 100 mM 0.71 mls 14.1 M stock from bottle

SDS 2% 20 mls 10% stock

62.5 mM Tris, pH 6.8 12.5 mls SDS-PAGE upper gel buffer

Wash blot well with aq, including warm water, add stripping solution warmed to 50oC in closed container, incubate 30 mins, decant with large volume of running water, wash again with aq, and put into Blotto to reblot. Would recommend putting back into ECL to check efficiency of strip, and repeating the secondary control to check for background. Note foreground goes down with every strip, and background comes up.

To strip membrane after immunoblot - Heat Detergent-Manue

Stripping buffer: 100 mM ß-mercapto-ethanol, 2% SDS, 62.5 mM Tris-Cl pH 6.8

- ß-mercaptoethanol 176 µl 342 µl

- 20% SDS 2.5 ml 5 ml

- Tris-Cl pH 6.7 1.56 ml 3.125 ml

- to H2O 25 ml 50 ml

Incubate and agitate the membrane in stripping buffer for 30 min. at 50°C

Wash 6 X for 5 min. minimum each in 1X PBS - 0.1% Tween

Optional:

Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent

Expose the film for 1 min. to 1 hr to make sure that the original signal is removed

Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween

The membrane is ready to reuse

Start at the blocking step

To strip membrane after immunoblot - Low pH-Manue

Stripping buffer: 25 mM glycine-HCl, pH 2, 1% SDS

- Glycine 1.876 g

- 20% SDS 50 ml

- to H2O 1 L

Incubate and agitate the membrane in stripping buffer for 30 min. at room temperature

Wash 3 X for 10 min. each in 1X PBS - 0.1% Tween

Optional:

Incubate the membrane for 1 min. in the Western Blot Chemiluminescence Reagent

Expose the film for 1 min. to 1 hr to make sure that the original signal is removed

Wash the membrane again 4 X for 5 min. minimum each in 1X PBS - 0.1% Tween

The membrane is ready to reuse

Start at the blocking step

Membrane: Protran (Schleicher and Schuell)

Rq: a PVDF membrane enables to sequence direcly from the membrane

Red ponceau S

Prepare a 2% (2 mg/ml) stock solution of Ponceau S dye in 30% (wt/v) trichloroacetic acid

Dilute the stock 10-fold in 1% acetic acid (final concentration of Ponceau S is 0.2%)

1X PBS - 0.1% Tween

- 5X PBS 200 ml

- Tween 20 1 ml

- to H2O 1 L

5% non-fat dry milk in 1X PBS - 0.1% Tween

- Non-fat dry milk 5 g

- to 1X PBS - 0.1% Tween 100 ml

Rq: this solution can be replaced by 3% BSA - 1X PBS - 0.2% Tween - sodiumazide (see Rodger)

Paint Pen Polymark: Natural Glow No. PM501, Dimensional Fabric and Graft