The captured images were digitized and the general cannabinoid receptor degrees compared after densitometry analysis. The primary antibody solutions were removed and blots cleaned as described previously. Extra antibody was added and incubated for 4 h, with shaking. The secondary antibody was removed and blots washed as described. Blots were incubated Docetaxel 114977-28-5 for 1 minute with equal volumes of ECL detection reagents 1 and 2. Chemiluminescence was taken for 2 h and saved as a TIFF file by a Flurochem 8900 MultiImage Light Cabinet. This antigen is similar to the corresponding sequence in canine, rat, murine and bovine species. The CB2 receptor polyclonal antibody was raised against amino acids 20 C33 in a sequence between the N terminus and the first transmembrane domain of the protein of the individual CB2 receptor. Human and murine CB2 Cholangiocarcinoma receptors demonstrate 82-year homology at the amino-acid level on the total protein. CB1 and CB2 blocking proteins were based on the CB1 and CB2 receptor sequences employed as antigens for creation of the respective polyclonal antiserum. Cannabinoid receptor binding Each binding analysis contained 30 g of back membrane protein in a final volume of 1 mL in binding buffer, as described previously. CP 55, 940 binds with equivalent affinity to CB1 and CB2 receptors with an approximate Ki of 0. 5 nmol/L. Particular CB1 receptor binding was thought as Aurora C inhibitor the binding of a receptor saturating concentration of CP 55, 940 displaced with a receptor saturating concentration of the CB1 particular ligand AM 251. AM 251 displays high affinity for CB1 receptors with a Ki value around 7 nmol/L, although its affinity at CB2 receptors has ended 300 flip weaker. Specific CB2 binding was defined as the binding of 5 nmol/L CP 55, 940 displaced with a receptor saturating concentration of the CB2 selective ligand AM 630. AM 630 binds CB2 receptors with high affinity, whereas its affinity for CB1 receptors is more than 165 fold less. All binding experiments were performed in triplicate. Reactions were terminated by quick vacuum filtration through Whatman GF/B glass fiber filters followed by two washes with ice-cold binding buffer. About 4 mL of Scintiverse was included with the filters and radioactivity quantified by scintillation counting. GTP S binding GTP S binding assays were performed as described previously in a buffer containing 20 mmol/L Hepes, 100 mmol/L NaCl, and 10 mmol/L MgCl2 at pH 7. 4. Each binding reaction contained 10 g of spinal cord membrane protein, the presence or lack of cannabinoid ligands, plus 0. 1 nmol/L 10 mol/L of GDP and GTP S to control basal G protein activation. Reactions were incubated for 2 h at 30 C. Non specific binding was defined by binding observed in the presence of 10 mol/L of non radioactive GTP S. In these reports, we first established the minimum concentration of the neutral CB1 antagonist O 2050 required to completely block CB1 mediated G protein activation by HU-210.

No related posts.