Western Blot Protocol(2)

Recipes
Lysate Buffer (Plasmid Prep Solution #1: 0.1 M NaCl, 50 mM Tris-HCl pH 7.8-8.5, 10 mM EDTA)
For 100 ml, add 2 ml of 5 M NaCl, 5 ml of 1 M Tris-HCl stock at the appropriate pH, 2 ml 0.5 M EDTA and Q.S. with distilled water. Autoclave.

5% Nonfat Dry Milk
Prepare using Carnation Nonfat Dry Milk in TBST.

10% Ammonium Persulfate (APS) Solution

0.12 g APS

1.2 ml H2O)
Keep for no more than week week in the refrigerator.

TBST (10 mM Tris-HCl, 0.15 M NaCl, 8 mM sodium azide, 0.05% tween-20, pH 8.0)

Chemical
For 4 liters
For 500 ml

Tris
4.84 g
0.61 g Tris

NaCl
35.06 g
4.38 g NaCl

NaN3
2.0 g
500 l 10% NaN3

Tween-20
2.0 ml
250 l Tween-20

Adjust pH to 8.0 with HCl. QS with water and store at 4C.

Tank Buffer (25 mM Tris-HCl, 0.2 M glycine, 0.35% SDS)

Chemical
For 4 liters
For 500 ml

Tris
12.11g
1.51 g Tris

Glycine
57.6 g
7.2 g

SDS
4.0 g
17.5 ml 10% SDS

QS with H2O. pH should be 8.3 but adjusting is not needed. Store at 4C

Transfer Buffers:

Wet blot transfer buffer (3L) (25 mM Tris-HCl, 0.2 M glycine, 20% methanol).
9.08 g Tris
43.24 g glycine
600 ml methanol
QS to 3 L with water.

Dry blot transfer buffers

Stock Solution A1 (3 M Tris)
36.3 g Tris, dissolved in double distilled water and QS to 100 ml
Store at room temperature.
Stock Solution A2 (0.3 M Tris)
3.63 g Tris, dissolved in double distilled water and QS to 100 ml
Store at 4C.
Stock Solution C (0.3 M Tris, 0.4 M aminohexane)
3.63 g Tris, 5.2 g aminohexane, dissolved in double distilled water and qs to to 100 ml.
Store at 4C.

Dry Blot Working Solutions (made from fry blot transfer buffers)
7 ml H20
2 ml Methanol
1 ml stock solution of A1, or A2 or C
Store at 4C.

Lower Gel Buffer (1.5 M Tris-HCl, 0.4% SDS, pH 8.8)
For 100 ml:
18.17 g Tris
4 ml 10% SDS
Adjust pH to 8.8 with HCl. QS to 100 ml with water. Store at room temperature.

Upper Gel Buffer 0.5 M Tris-HCl, 0.4% SDS, pH 6.8)
For 100 ml:
6.06 g Tris
4 ml 10% SDS
Adjust pH to 6.8 with HCl. QS to 100 ml with water. Store at room temperature.

4x Sample Buffer (made from stock sample buffer) (0.24 M Tris-HCl, 0.24 M SDS, 40% glycerol, 20% 2-mercaptoethanol, pH 6.8)
8 ml of stock sample buffer
2 ml of concentrated 2-mercaptoethanol
Transfer to 1 ml aliquots and store at -20C.

2x Sample Buffer (Hoeffer Scientific) (125 mM Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue).
Prepare in fume hood!
2.5 ml 0.5 M Tris-HCl, pH6.8 and 4.0 ml of 10% SDS
(--or-- 2.5 ml of upper gel buffer and 3.6 ml of 10% SDS)
2.0 ml glycerol
1.0 ml concentrated 2-mercaptoethanol
0.4 mg bromophenol blue (you may use less but don't add more than 0.4 mg)
QS to 10 ml with water. Transfer to 1 ml aliquots and store at -20C.

Stock Sample Buffer (from Craig Wasson) (0.312 M Tris-HCl, 0.346 M SDS, 50% glycerol, pH 6.8)
3.03 g Tris
8.0 g SDS
40 ml glycerol
Adjust pH to 6.8 with HCl. QS to 80 ml with water. Store at room temperature.

0.1% Fast Green (in 10% acetic acid and 20% methanol)
0.2 g Fast Green
20 ml acetic acid
40 ml methanol
140 ml double distilled water.

0.1% Amido Black (in 10% acetic acid and 45% methanol)
0.2 g Buffalo Black
90 ml methanol
20 ml acetic acid
90 ml double distilled water.

Amido Black Destain (2% acetic acid, 45% methanol)
225 ml methanol
10 ml acetic acid
QS to 500 ml with double distilled water.

Protein Molecular Weight Markers
We use Promega and Pharmacia molecular weight markers.

Promega Mid-Range Marker
Pharmacia LMW Markers

Phosphorylase B 97.4 kD
Phosphorylase B 94 kD

BSA 66.2 kD
BSA 67 kD

L-glutamate dehydrogenase 55 kD

Ovalbumin 42.7 kd
Ovalbumin 43 kD

Aldolase 40 kD

Carbonic anhydrase 31kD
Carbonic anhydrase 30 kD

Soybean trypsin inhibitor 21.5 kD
Soybean trypsin inhibitor 22 kD

Lysozyme 14.3 kd
a-Lactalbumin 14.4 kD

Supplies

Tips

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Western Blot Protocol(2)