protocol-GPCR-WB

Western Blot Protocol (for use with GPCRs antibodies) Buffers to prepare: SDS-PAGE sample buffer: 2x Sample Buffer Volume Material 3.55 mL Demonized water 1.25 mL 0.5 M Tris-HCl, pH 6,8 2.5 mL Glycerol 2.0 mL 10 % (w/v) SDS 0.2 mL 0.5 % (w/v) bromophenol blue 9.5 mL 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 Tris base 144.0 g Glycin 10.0 g 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 µg per sample (for total brain membranes 50 µg is recommended). Mix equal quantities of sample buffer and sample and heat for 5 min at 65 oC. Polyacrilamide Gel (8%): Upper (4%): Reagent Volume Tris 0.5M, pH 6.8 2.5 mL SDS 10% 100 µL Acrilamida 40% / Bis 1% 1.0 mL APS 10% 50 µL TEMED 10 µL Water 6.40 mL TOTAL 10 mL Lower (8%): Reagents Volume Tris 1.5M, pH 8.8 2.5 mL SDS 10% 200 µL Acrilamida 40% / Bis 1% 2.0 mL APS 10% 50 µL TEMED 5 µL Water 5.6 mL TOTAL 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 end- user. 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 manufacturer’s 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.