Required Solutions

10x PBS

80 g NaCl

2 g KCl

14.4 g Na2HPO4

2.4 g KH2PO4

Dissolve into 800 ml ddH2O

Adjust pH to 7.4

Qs to 1000 ml

Autoclave

Make a 1x working solution

Buffer C (4ºC)

20 mM Hepes-KOH pH 7.9 (from 0.5 M stock)

0.42 M KCl (from 3 M stock)

25% glycerol (from 80% stock)

0.1 mM EDTA (from 0.5 M stock pH 8.0)

5 mM MgCl2 (from 1 M stock)

0.2% NP40 (from 10% stock)

Use autoclaved stocks for all ingredients.

Store at 4ºC

Add right before use to 10 ml:

10 µl 1 M DTT  (store aliquots at -20ºC, thaw once)

50 µl 100 mM PMSF (in iso-propanol -20ºC)

1 µl 10 mg/ml Leupeptin (-20ºC)

1 µl 10 mg/ml Aprotinin (-20ºC)

10 µl 1 mg/ml Pepstatin (-20ºC)

Or, replace the last three protease inhibitors with one mini Roche protease inhibitor

Tablet (# 11 836 153001) and add DTT and PMSF as above.

For looking at phosphorylated proteins, add the following phosphatase inhibitors to

Buffer C:

Na-beta-glycerophosphate to 50 mM (stock 1 M)

NaF to 1 mM (stock 0.4 M)

Na-ortho-vanadate to 1 mM (stock 0.1 M)

Buffer D

20 mM Hepes-KOH pH 7.9

100 mM KCl

25% glycerol

0.1 mM EDTA (cold)

Add right before use, for 250 ml:

250 µl 1 M DTT

1.25 ml 100 mM PMSF

10x Ponceau S

Ponceau S 2% (w/v)

TCA 30% (w/v)

Dilute to 1x before use (can be re-used many times).

4x Laemmli Buffer

4.4 ml 0.5 M Tris (pH 6.8)

4.4 ml Glycerol

2.2 ml 20% SDS

0.5 ml 1% Bromophenol Blue

0.5 ml Beta-ME

Aliquot and store at -20ºC. Dilute to 2x before use.

10x Running Buffer

30.3 g (0.25 M) Tris Base

144 g (1.92 M) Glycine

10 g (1%) SDS or appropriate for concentrated stock

Qs 1000 ml ddH2O

Dilute 1:10 with ddH2O.  PH will be 8.3

10x Towbin’s Electrotransfer Buffer

30.3 g Tris Base

144 g Glycine

Qs 1000 ml ddH2O

1x Towbin’s

100 ml 10x stock

200 ml (20%) Methanol

Qs 1000 ml ddH2O

1 ml (0.02%) 20% SDS- optional