Western Blotting for CB1 Aurélie, 2006
Western Blotting for CB1 antibody
I. Preparation of the antibody from alpha diagnostic
Reconstitution: Add 100 µl of mQW in 100 µg AB or BP
Ä Turn on bath
Ä Defreeze AB
Incubation: Dilution 1/500, so 30 µl of AB1 in 15 ml
1) Incubate at 37˚ for 1.5 h
2) Incubate at 4˚ for 2-24 h
3) Centrifuge at 10,000 rpm at 4˚ for 15 min
4) Put the supernatant in 15 µl of AIB
II. Preparation of Gels (for 2 gels)
Ä Defreeze APS
- Plug:
- Resolving gel (takes up 3/4 of plate) and after add a layer of butanol
- Wash of butanol with MilliQ before adding stacking gel (1/4 of plate).
Plug / Resolving gel / Stacking gel / Plug x2 / Resolving gel x2 / Stacking gel x2Acrylamide / 0.75 ml / 2.5ml / 0.5 ml / 1.5 ml / 5 ml / 1 ml
Resolving gel (1M) / 3.75 ml / 7.5 ml
Stacking gel / 1.25 ml / 2.5 ml
mQW / 1.25 ml / 3.55 ml / 3 ml / 2.5 ml / 7.1 ml / 6 ml
10% SDS / 100 µl / 50 µl / 200 µl / 100 µl
APS (40%) / 20 µl / 50 µl / 25 µL / 40 µl / 100 µl / 50 µl
TEMED / 1.25 µl / 5 µL / 2 µL / 2.5 µl / 10 µl / 4 µl
III. Electrophoresis
Electrophoresis Buffer: Tris-HCl (PH=8.3):
28.82 g of Glycine 100 mL of 1M Tris
50 mL of Tris-HCl (pH 8.3) 39.8 mL of 1M HCl
20 mL of 10% SDS
à 2 L of mQW (can be recycled once)
Ä Put gels face inside!
- Leave protein samples in buffer for 15 min
- Boil the samples before loading in hot plate: 70 °C, 5 min
- Load 20µg in 15 µL of each diluted (in sample buffer) sample in each lane.
- Use 15 µL of the Pre-Stain molecular weight (high MW) markers
- Add 15 µL of straight sample buffer to empty lanes.
- Gel is run at 125V constant voltage for 60 min at room temperature.
IV. Transfer (Western Blotting)
Transfer Buffer: 10x Transfer Buffer:
100 mL of 10x Transfer Buffer 22.13 g of CAPS
100 mL of Methanol à 1 L with mQW
à 1 L with mQW
PH: Add 2 Sodium Hydroxide Pellet (pH =11)
- PVDF membrane: 8.8 cm x 7.5 cm (don’t forget to mark it)
- Soak the membrane in 100% Methanol for 15 minutes to activate
- Soak filter paper, foam, and PVDF membrane in transfer buffer
- Black – Foam – Filter – Gel – Membrane – Filter – Foam – White
- Remove air bubbles
- Transfer for 120 min at 280mA constant current on ice.
V. Blocking
Blocking Solution: 4 g of Milk in 40 mL of Washing Buffer (for 2 Gels)
- Membrane is left covered in blocking solution 1 hour (shaking at RT).
VI. Antibody Incubation
Antibody Incubation Buffer: 2 g of Milk in 40 mL of Washing Buffer (for 2 Gels)
10x Washing Buffer: 1 L of 1M Tris-HCl 1M Tris-HCl:
175.3 g of NaCl 456.5 mL of 1M HCl
36.2 mL of Tween 20 543.5 mL of 1M Tris
à 2 L with mQW
1M HCl:
Stock at 11.8M ð 85 mL of Stock in 1 L
- Rinse the membrane with antibody incubation buffer (AIB) once (4 mL).
- The primary antibody CB1 Rabbit 1/500 (30 µL) is added to PVDF soaked in 15 ml of the antibody incubation solution and left shaking at 4° overnight.
SECOND DAY:
Ä Defreeze antibody II
- 3x 5min and 4x quick washes in washing buffer, and rinse with AIB once (4 mL).
- The secondary antibody anti Rabbit 1/5 000 (3 µL) is added to PVDF soaked in 15ml of the antibody incubation buffer and left shaking at RT for 1 hour.
- 3x 5min and 4x quick washes in washing buffer, and a final wash in mQW.
VII. Band analysis by Densitometry by Chemiluminescence (elkus_1807)
- West Dura chemiluminescent substrate: 1mL A + 1mL B for 2 Blots
- Exposure on the KODAK imaging station: 30-40 min
- CB1: 60 kDA (3rd band of Marker)
VIII. Stripping of the membrane
- 5X Tris solution at 0.3152 M (38.1392 g/L), pH 6.8. Add 10g of SDS per 100ml of 5X Tris.
- Dilute to 1X with mQW, and add 0.35 mL β-mercaptoEtOH per 50 mL of 1X Tris.
- Incubate at 50°C for 30 min
- 5 washes
- Reblock the membrane if use of neurofilament
IX. Proteins detection with SYPRO
- Allow the membrane to dry completely
- Float the mb face down in 7% acetic acid, 10% methanol for 15 min
- 4 washes of 5 min with mQW
- Incubate with SYPRO for 15 min (SYPRO can be re-utilized)
- 2-3 washes of 1 min in mQW
- Allow the membrane to dry completely
- Imaging with UV at 590 nm (don’t forget to switch off UV after use)
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