Griffin:Antibody Related Solutions & Recipes

Acrylamide Stock Solution

To make 1L:

• 30% Acrylamide: 300 g Acrylamide
• 0.8% Bis-Acrylamide: 8.0 g Bis-Acrylamide

Materials:

• Acrylamide (crystalline; store @ 4C)
• Bisacrylamide (crystalline; store at room temperature)
• Filter Unit (Nalgene 500 ml 0.2 micron disposable filter unit)
• Light proof container

Methods: To make 500 ml Stock Solution

I) Place 300 ml ddH20 in beaker with stir bar

II) Add 150.0 g acrylamide to the H20 while stirring

III) Add 4.0 g bisacrylamide to the H20 while stirring

IV) Allow the acrylamide & bisacrylamide to dissolve

V) Bring the final volume to 500 ml

VI) Vacuum filter the acrylamide/bisacrylamide solution

VII) Place the acrylamide stock solution in a light proof container @ 4C

Blocking Buffer (Bovine Serum Albumin)

To make 1L:

• 20 mM Tris: 2.42 g Tris Base pH 7.4 w/HCl
• 150 mM NaCl: 8.76 g NaCl
• 0.01% Tween-20: 0.1 ml Tween-20
• 3% BSA: 30.0 g BSA (Fraction V)
• 0.02% NaN3: 10.0 ml 2% NaN3

Blocking Buffer (milk)

To make 1L:

• 20 mM Tris: 2.42 g Tris Base pH 7.4 w/HCl
• 150 mM NaCl: 8.76 g NaCl
• 0.01% Tween-20: 0.1 ml Tween-20
• 5% Milk: 50.0 g Nonfat Dry Milk
• 0.02% NaN3: 10.0 ml 2% NaN3

NET-G buffer

• 150 mM NaCl
• 5 mM EDTA
• 50 mM Tris
• 0.05% Triton X-100
• 0.25% Gelatin
• pH 7.4

Resolving Gel Buffer

To make 1L:

• 0.75 M Tris pH 8.9: 90.8 g Tris Base pH 8.9 w HCl
• 4 mM EDTA: 8.0 ml of 0.5 M EDTA (tetra Na is the most soluble)
• 0.2% SDS: 20.0 ml 10% SDS

Running Buffer

To make 1L:

• 50 mM Tris: 6.06 g Tris Base
• 380 mM Glycine: 28.5 g Glycine
• 1.6 mM EDTA: 0.67 g EDTA (tetra Na is the most soluble)
• 0.1% SDS: 1.0 g SDS

Stacking Gel Buffer

To make 1L:

• 0.1 M Tris pH6.7: 12.2 g Tris Base pH 6.7 w/H3PO4
• 4 mM EDTA: 8.0 ml of 0.5 M EDTA (tetra Na is the most soluble)
• 0.2% SDS: 20 ml 10% SDS

Stripping Buffer

To make 1L:

• 62.5 mM Tris pH 6.8: 7.0 g Tris base ph 6.8 w/HCl
• 100 mM 2-Mercaptoethanol: 7.0 ml 2-Mercaptoethanol (14.3 mol/L Stock)
• 2% SDS: 20.0 g SDS

Transfer Buffer

To make 1L:

• 25 mM Tris: 3.03 g Tris Base
• 192 mM Glycine: 14.41 g Glycine
• 0.02% SDS: 0.2 g SDS
• 20% Methanol: 200 ml Methanol

Tris Buffer Saline (TBS Tween, Wash Buffer)

To make 1L:

• 20 mM Tris: 2.42 g Tris Base pH 7.4 w/ HCl
• 150 mM NaCl: 8.76 g NaCl
• 0.01% Tween-20: 0.1 ml Tween-20 and and