30 Jun 2011

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

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