Griffin:Antibody Related Solutions & Recipes
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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