Western Blot Recipes

PBS 10X

• 40 g NaCl
• 1 g KCl
• 13.4 g Na₂HPO₄-7H₂O
• 1.2 g H₂O

Adjust the pH at 7.4 with HCl. Complete to 500 mL with distilled water.

Lysis Buffer

• 580 µL Nonidet P40
• 0.58 g CHAPS
• 0.150 g HEPES
• 0.508 g NaCl
• 9.93 g Saccharose
• 0.022 g Na₂EDTA

Adjust the pH to 8.0. Complete to 50 mL with distilled water.

Laemmli buffer samples 5X

• 1 mL Glycerol
• 1 g SDS 10%
• 6.25 mL Tris HCl 0.5M pH 6.8
• 2.5 mL β-mercaptoethanol
• 1 mL Bromophenol 0.5%

 Complete to 10 mL with distilled water.

Acrylis bis 30%

• 29.2 g Acrylamide (29.2%)
• 0.8 g N-N’-methylene-bisacrylamide (0.8%)

Complete to 100 mL with distilled water.

SDS 10%

• 100 g Sodium Dodecyl Sulfate
• 1000 mL distilled H₂O

PS 10%

• 1 g Ammonium Persulfate
• 10 mL distilled H₂O

Prepare it fresh.

Running Buffer 1.5 M, pH 8.8

• 90.75 g Tris
• 500 mL distilled H₂O

Adjust the pH at 8.8 with HCl.

Stacking Buffer 0.5 M, pH 6.8

• 6 g Tris
• 100 mL distilled H₂O

Adjust the pH at 6.8 with HCl.

TANK Buffer 10X

• 15 g Tris
• 72 g Glycine
• 50 mL SDS 10%

Heat for ???

Complete to 500 mL with distilled water.

TANK Buffer 1X

• 100 mL Tank 10X
• 900 mL distilled H₂O

Transfer buffer 1X

• 3.03 g Tris HCl
• 14.4 g Glycine
• 200 mL Methanol
• 1 mL SDS 10%

Complete to 1 liter with distilled water.

TBS 10X

• 24.2 g Tris
• 84 g NaCl

Adlust the pH to 7.6 with HCl.

Complete to 1 liter with distilled water.

T-TBS 1X

To 1 liter of TBS 1X, add 1 mL of Tween 20.

Blocking Buffer

To 10 mL of T-TBS 1X, add 0.5 g of non fat dry milk.

Coomassie blue R-250 0.5%

• 0.5 g Coomassie blue R-250 (Biorad, 161-0400)
• 40 mL Methanol
• 10 mL Acetic acid

Complete to 100 mL with distilled water. Shaking with magnet for some hours. It is possible to recuperate the dye for 3-4 gels.

Decoloration solution

• 25 mL Methanol
• 50 mL Glacial acetic acid

Complete to 1 liter with distilled water.

Stripping Buffer

• 10 mL β-mercaptoethanol
• 200 mL SDS 10%
• 137 mL Tris-HCl 0.5 M, pH 6.8

Complete to 1 liter with distilled water.