7TM Western Blot Protocol

1. Buffers and Reagents

Use double distilled water for buffer preparation or water with the same grade of purity.

• Blocking buffer: TBST with 5% BSA (bovine serum albumin)
• HA-beads: Agarose beads coupled with anti-HA-tag antibody (only for immunoprecipitation of HA-tagged GPCR receptors)
• Inhibitors: Protease inhibitors (PMSF 100µM, Leupeptin 10µg/ml, Aprotinin 5µg/ml, Pepstatin A 1µg/ml). Alternatively use protease inhibitor tablets. Phosphatase inhibitors: Use phosphatase inhibitor tablets (e.g. PhosSTOP™ from Roche)
• Lysis buffer: 150 mM NaCl, 50 mM Tris-HCl, 5mM EDTA, 1% Igepal (Nonidet P-40), 0.5% Na-Deoxycholat, 0.1% SDS
• Poly-L-lysine: 0.1 mg/ml
• PBS: Dulbecco’s Phosphate Buffered Saline (NaCl: 137 mM, Na2HPO4: 8.1 mM, KH2PO4: 1.47 mM, KCl: 2.68 mM, pH 7.4)
• SDS-Sample-buffer: 62.5 mM Tris (pH 7.6), 2% SDS, 20% Glycerol, 100 mM Dithiothreitol (DTT), 0.005% Bromophenol Blue
• TBS: Tris Buffered Saline (Tris: 0.05 mM, NaCl: 150 mM, pH 7.6)
• TBST: TBS with 0.1% Tween 20
• WGA-beads: Wheat germ agglutinin lectin conjugated to agarose-beads, (for easy enrichment of glycosylated proteins including nearly all GPCR family members from cell and tissue extracts)

2. Sample Preparation, SDS-Gel and Blotting

1. Coat 60-mm dishes with poly-L-lysine for 30 min at room temperature. Aspirate poly-L-lysine. Wash 3-times with water. Aspirate water after each step. Dry dishes for 30 min at room temperature.
2. Seed cells into 60-mm dishes and let them grow to 80% confluence.
3. Treat cells for desired time with or without compound (e.g. agonist, antagonist or inhibitor) in fresh media.
4. Aspirate media. Wash with ice-cold PBS. Aspirate PBS.
5. Apply 800 µl ice-cold lysis buffer (with inhibitors) and incubate on ice for 10 min.
6. Harvest cells with a cell scraper and transfer extract to a 1.5 ml microcentrifuge tube.
7.  Centrifuge for 30 min at 14.000 x g and 4°C.
8. Transfer 30-50 µl WGA- or 30 µl HA-beads to 1.5 ml microcentrifuge tubes and wash once with ice-cold lysis buffer (without Inhibitors). Aspirate lysis buffer.
9. Apply supernatant of (7) and rotate for 1-2 h on a rotation shaker at 4°C.
10.  Wash 3 times with ice-cold lysis buffer (centrifuge shortly at 4°C). Aspirate lysis buffer.
11. Remove remaining lysis buffer with an insulin syringe, add 50-100 µl 1 x SDS-Sample-buffer (including DTT) and start elution (elution temperature and time varies according to receptor and glycosylation status, we recommend to start with 20 min elution at 50 °C) Do not use temperatures above 60°C!
12. Centrifuge shortly (5 min full speed)
13. Load 20 µl supernatant onto SDS-PAGE (we recommend use of prestained molecular weight markers to verify transfer and determination of molecular weights).
14. Electrotransfer blotting to PVDF or nitrocellulose membrane.

3. Blocking, Antibody incubation and Detection

1. Incubate blot for 1 hour in blocking solution with gentle agitation. The use of other or commercially available blocking solutions is also suitable.
2. Incubate blot with Premium 7TM Antibodies at a dilution of 1:1000 in blocking solution for 2 hours at room temperature or at 4°C overnight with gentle agitation.
3. Wash blot with TBST for 10 min with gentle agitation. Aspirate TBST. Repeat 3 times.
4. Incubate blot with anti-rabbit HRP-coupled secondary antibody in blocking solution for 2 hours at room temperature or at 4°C overnight with gentle agitation.
5. Wash blot with TBST for 10 min with gentle agitation. Aspirate TBST. Repeat 3 times.
6. Incubate blot with HRP-substrate. We recommend commercially available HRP-substrates.
7. Detection of chemiluminescence via X-ray film or chemiluminescence detection system of your choice.

For more information please contact us:

E-Mail:       support@7tmantibodies.com
Fon:           0049-151-20130575
FAX:           0049-3641-2414958