Western Blot Recipes
Western Blot Lower Gel Buffer (WB-LGB)
Store in dark bottle at room temperature
| 1 Gel | 2 Gels | 3 Gels |
| 10 mL Acrylamide | 20 mL Acrylamide | 30 mL Acrylamide |
| 12.4 mL ddH2O | 24.8 mL ddH2O | 37.2 mL ddH2O |
| 7.5 mL LGB (4x) | 15.0 mL LGB (4x) | 22.5 mL UGB (4x) |
| 10 uL TEMED | 20 uL TEMED | 30 uL TEMED |
| 200 uL APS | 400 uL APS | 600 uL APS |
Vortex first three ingredients, then add APS and TEMED.
APS (Ammonium Persulfate) 12% Stock
57 mg. APS into 475 uL ddH2O (10%)
Western Blot Upper Gel Buffer (WB-UGB)
| 1 Gel | 2 Gels | 3 Gels |
| 2.7 mL Acrylamide | 5.4 mL Acrylamide | 8.1 mL Acrylamide |
| 12.1 mL ddH2O | 24.2 mL ddH2O | 36.3 mL ddH2O |
| 5 mL UGB | 10 mL UGB | 15 mL UGB |
| 10 uL TEMED | 20 uL TEMED | 30 uL TEMED |
| 200 uL APS | 400 uL APS | 600 uL APS |
12% Gel:
- 12 mL Acrylamide
- 10.4 mL ddH2O
- 7.5 mL LGB
20x TBS
48.44 g. Tris (pH 7.4)
151.9 g. NaCl
700 mL H2O
Adjust pH to 7.4
q.s. to 1 liter
1x PBS Blocking Buffer
- 10 mLs of 45% fish gelatin
- 100 mLs of 10x PBS
- 0.2 g. Sodium Azide (NaN3)
- 1.0 g of casein
- 888.8 mL of ddH2O
Filter sterilize through a 0.45 uM filter
PBS-Tween
- 9000 mL PBS
- 2.0 mL Tweene
- q.s. 1L H2O
10x Running Buffer
- 30.3g tris base (trizma)
- 144.1g glycine
- 10g SDS
- q.s. to 1000ml with dd H2O
- pH 8.3
1x Running Buffer
- dilute 10x with ddH2O
10x Transfer Buffer
- 30.3g tris base (trizma)
- 144.1g glycine
- q.s. to 1000ml with ddH2O
- pH 8.3
1x Transfer Buffer
- 100ml 10x Transfer Buffer
- 500ml ddH2O
- 200ml methanol
- q.s. to 1000ml with ddH2O
SDS Sample Buffer, 5x
- 1.56 mL 2M Tris pH 6.8
- 2.5 mL BME
- 5.0 mL Glycerol
- 1.0 g. SDS
- 0.0005 g. Bromophenol Blue
- q.s. to 10 mL w/ ddH2O
Tris, 2M, pH 6.8
- 150 mL H2O
- 60.55 g. Tris
- Adjust to pH 6.8 w/ HCl (will take a lot… ~75-100 mL)
- q.s. to 250 mL w/ H2O
SDS Sample Buffer Stock
- 15.6 mL 2M Tris (pH 6.8)
- 25 mL BME
- 50 mL Glycerol
- 10 g. SDS
- 0.005 g. Bromophenol Blue
- q.s. to 100 mL w/ ddH2O
Antibody 12CA 519
1:500 dilution:
- 10 mL milk
- 20 uL 12CA 519
Antibody Rabbit Anti Mouse
1:1000 dilution:
- 10 mL milk
- 10 uL Rabbit Anti Mouse
1:500 dilution:
- 10 mL milk
- 20 uL Rabbit Anti Mouse
I-125
1:1000 dilution
- 30 uL I-125
- 30 mL milk
1 M CAPS, pH 11.0 (N-cyclohexyl-3-aminopropanesulfonic acid)
- 221.3 g. CAPS
- 40 g. NaOH
- q.s. to 1 L
*Check pH; (should be 11.0) may need to alter slightly.