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
 Turn on bath
 Defreeze AB
Incubation: Dilution 1/500, so 30 µl of AB1 in 15 ml
1)
2)
3)
4)
Incubate at 37˚ for 1.5 h
Incubate at 4˚ for 2-24 h
Centrifuge at 10,000 rpm at 4˚ for 15 min
Put the supernatant in 15 µl of AIB
30 µl CB1
100 µl PBS
120 µl mQW
30 µl CB1
100 µl PBS
120 µl BP
II. Preparation of Gels (for 2 gels)
 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).
Acrylamide
Plug
Resolving
gel
Stacking
gel
0.75
ml
2.5ml
0.5 ml
Resolving gel
(1M)
Stacking gel
mQW
10% SDS
APS (40%)
TEMED
Plug
x2
1.5
ml
3.75 ml
Resolving gel
x2
Stacking gel
x2
5 ml
1 ml
7.5 ml
1.25 ml
1.25
ml
20 µl
1.25
µl
3.55 ml
3 ml
100 µl
50 µl
50 µl
25 µL
5 µL
2 µL
2.5 ml
2.5
ml
7.1 ml
6 ml
40 µl
200 µl
100 µl
100 µl
50 µl
2.5 µl
10 µl
4 µl
III. Electrophoresis
Electrophoresis Buffer:
28.82 g of Glycine
50 mL of Tris-HCl (pH 8.3)
20 mL of 10% SDS
 2 L of mQW (can be recycled once)
 Put
-
Tris-HCl (PH=8.3):
100 mL of 1M Tris
39.8 mL of 1M HCl
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.
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Western Blotting for CB1
Aurélie, 2006
IV. Transfer (Western Blotting)
Transfer Buffer:
100 mL of 10x Transfer Buffer
100 mL of Methanol
 1 L with mQW
PH: Add 2 Sodium Hydroxide Pellet (pH =11)
-
10x Transfer Buffer:
22.13 g of CAPS
 1 L with mQW
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
175.3 g of NaCl
36.2 mL of Tween 20
 2 L with mQW
1M Tris-HCl:
456.5 mL of 1M HCl
543.5 mL of 1M Tris
1M HCl:
Stock at 11.8M  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:
 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
2
Western Blotting for CB1
Aurélie, 2006
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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