Western Blotting for CB1 Antibody

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).

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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