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 L of -Mercaptoethanol to 950 L 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.

SDS-PAGE Transfer Buffer: 1. 25 mM Tris-Base 2. 192 mM Glycin 3. 20% Methanol 4. pH 8.3 For 1 L of buffer mix 3.03 g of Tris-Base, 14.4 g of glycin and 200 mL of methanol; Bring to 1L with deionized water. Do not pH. Western Blot Protocol: The amount of protein that should be loaded to the gel varies with the experiment; it can be 25 to 100 g per sample (for total brain membranes 50 g is recommended). Mix equal quantities of sample buffer and sample and heat for 5 min at 65 oC. Polyacrilamide Gel (8%): Upper (4%): Reagent Tris 0.5M, pH 6.8 SDS 10% Acrilamida 40% / Bis 1% APS 10% TEMED Water TOTAL Lower (8%): Reagents Tris 1.5M, pH 8.8 SDS 10% Acrilamida 40% / Bis 1% APS 10% TEMED Volume 2.5 mL 200 L 2.0 mL 50 L 5 L Volume 2.5 mL 100 L 1.0 mL 50 L 10 L 6.40 mL 10 mL

Water TOTAL

5.6 mL 10 mL

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