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Western Blot Protocol

(for use with GPCRs antibodies)
Buffers to prepare: SDS-PAGE sample buffer: 2x Sample Buffer Volume 3.55 mL 1.25 mL 2.5 mL 2.0 mL 0.2 mL 9.5 mL Material Demonized water 0.5 M Tris-HCl, pH 6,8 Glycerol 10 % (w/v) SDS 0.5 % (w/v) bromophenol blue Total Volume

Add 50 µL of β-Mercaptoethanol to 950 µL of sample buffer before used. Mix the sample to the sample buffer at least 1:2 proportion and heat to a 95oC for 4 min. SDS-PAGE Running Buffer: 10x Running Buffer Weight Material 30.3 g 144.0 g 10.0 g Tris base Glycin SDS

Dissolve in deionized water and fill to 1000 mL. Store at 4 oC. Warm the buffer before use to remove possible precipitates. Do not pH.

Polyacrilamide Gel (8%): Upper (4%): Reagent Tris 0.0 mL 50 µL 5 µL Volume 2.5 mL 100 µL 1. 20% Methanol 4. 14.8 SDS 10% Acrilamida 40% / Bis 1% APS 10% TEMED Volume 2.0 mL 50 µL 10 µL 6. pH 8.40 mL 10 mL .8 SDS 10% Acrilamida 40% / Bis 1% APS 10% TEMED Water TOTAL Lower (8%): Reagents Tris 1. it can be 25 to 100 µg per sample (for total brain membranes 50 µg is recommended). Do not pH.5M. Bring to 1L with deionized water. Western Blot Protocol: The amount of protein that should be loaded to the gel varies with the experiment.5M.5 mL 200 µL 2. pH 8.3 For 1 L of buffer mix 3. Mix equal quantities of sample buffer and sample and heat for 5 min at 65 oC.SDS-PAGE Transfer Buffer: 1. 25 mM Tris-Base 2.4 g of glycin and 200 mL of methanol.03 g of Tris-Base. 192 mM Glycin 3. pH 6.

The GPCRs band should be in the middle of the gel. .1% Tween with the secondary antibody (Licor 760 nm anti-Rabbit) concentration 1:10 000 (or according to the manufacturer’s protocol). Secondary Antibody: Incubate for 2 hours in 1x PBS + 0.1% Tween). Primary Antibody: After blocking.1% Tween and them wash 3-4 times (20 min with 1x PBS + 0.Water TOTAL 5. See the product-specific specification sheet for recommended starting dilutions. Note that the optimal dilution for a specific antibody will vary and should be determined by the enduser.6 mL 10 mL After putting the sample in the gel. Blocking: Block with either 1x PBS + 3% Albumin or with 5% milk in PBS. Transfer: The transfer should be semi-dry for 40 min at 10 volts or wet overnight at 15 V / 90 mA. If there still some dirtiness wash an additional 3 times with 1x PBS + 0. Second wash: After the secondary incubation wash rapidly 3 times with 1x PBS + 0. without washing. wash 3 times with PBS and scan in the Licor. run until the blue marker goes to the end of the gel. put the membrane in 1x PBS with the anti-GPCR antibody in a 1:6000 dilution and incubate it "overnight" at 4oC in the shaker. Block for 4-6 hours shaking (slowly) at room temperature. First wash: After the primary antibody incubation wash the blot three times with 1x PBS and then wash 3 times (10 min with 1x PBS + 0. but it is important to run a marker together.1% Tween. Develop: Before developing the blot.1% Tween).