Immunoblotting of Cannabinoid Receptors

3.5.1. Protein Extraction From Blood or Tissue Samples

1. Blood or tissue samples are obtained for CBr protein determinations from the subjects.

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Fig. 4. [3H]Anandamide uptake by CHO cells expressing hDAT (black bar) and two unidentified transporters (P16, slashed bar and G25, hatched bar). Uptake was carried out for 5 min at 37°C in KRH buffer (see Subheading 3.). These CHO cells are stable cell lines. Data are mean ± SEM from three experiments.

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hDAT P16 G25

CHO Cell Genotype

Fig. 4. [3H]Anandamide uptake by CHO cells expressing hDAT (black bar) and two unidentified transporters (P16, slashed bar and G25, hatched bar). Uptake was carried out for 5 min at 37°C in KRH buffer (see Subheading 3.). These CHO cells are stable cell lines. Data are mean ± SEM from three experiments.

2. Two mL of blood or tissue sample was centrifuged at 5000 rpm at 4°C for 5 min and the supernatant discarded.

3. The pellet was resuspended in 400 |L of lysis buffer containing protease inhibitors and homogenized. After rinsing with 200 |L lysis buffer into a tube, the homogenate was centrifuged at 15000 rpm at 4°C for 15 min. The supernatant can be aliquoted and stored at -80°C for immunoblotting.

4. Samples are then prepared for immunoblotting using 50 |L of the homogenate and 2X Laemilli's buffer and boiled for 10 min. After cooling, the mixture was centrifuged at 1500 rpm at 4°C and the supernatant used for Western blotting.

3.5.2. Western Blotting

1. Equal amounts of protein were loaded and separated by 10% SDS-PAGE and then transferred to nitrocellulose. The blots were blocked in freshly prepared PBS containing 3% nonfat milk for 20 min at 20-25°C with constant agitation.

2. After incubation with a rabbit-anti-human CB1 antibody (Calbiochem, Cambridge, MA) for overnight at 4°C, a goat anti-rabbit IgG linked to horseradish peroxidase (Amersham Life Science products, Arlington Heights, IL) was added for an additional 1.5 h.

3. Blots are developed using enhanced chemiluminescence (Amersham Life Science products, ArlingtonHeights, IL).

4. Films are scanned in Pharmacia LKB Ultroscan XL enhanced laser densitometer and the results reported in relative units. (See Notes 2 and 3 for additional discussions on localization of CBrs and Fig. 5.)

Fig. 5. CB1r immunoblots from whole brains of mice following chronic treatment with vehicle A, metanandamide B (10 mg/kg), and rimonabant C (3 mg/kg). Each lane represents a pooled sample of six mice.

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