The endogenous cannabinoid system (ECS) controls a number of physiological and pathological conditions. They consist of the cannabinoid receptor type 1 (CB1) and type 2 (CB2), their endogenous ligands (endocannabinoids), and proteins that regulate endocannabinoid biosynthesis and degradation. CB1 is highly expressed in the brain tissue and CB2 is expressed exclusively in the cells of the immune system. G protein-coupled receptor 55 (GPR55) is a cannabinoid receptor that is, based on the amino acid sequence, less than 15 % similar to CB1 and CB2. The ability to bind various endogenous and exogenous cannabinoid ligands is the reason why GPR55 is a prime candidate for categorization as a cannabinoid receptor type 3. It is expressed in the brain tissue, endothelial cells and the digestive tract.
Within this diploma thesis, we wanted to determine the appropriate working concentrations of primary and secondary antibodies against CB1, CB2 and GPR55 using the dot blot method. In addition, for further quantitative determination of CB, we wanted to determine the suitability of antibodies from two manufacturers, Santa Cruz Biotechnology and Abcam, and selected antibodies from that manufacturer, with which we obtained better detection results. For this purpose, cell lysates were prepared from the selected cell line and the protein concentration of the soluble fraction of the cell lysate was measured with a set of bovine serum albumin (BSA) standards. Samples applied to the membrane for immunodetection were incubated in selected antibodies and then imaged with a LAS 4000 camera, which has the ability to capture chemiluminescent light. Our results suggest that antibodies against CB1 and CB2 from both manufacturers are appropriate. We decided that the antibodies manufactured by Santa Cruz Biotechnology were more suitable for the immunodetection of CB1 and CB2, in comparison with antibodies manufactured by Abcam, and as such were appropriate for further use in the quantitative determination of CB using the Western method. We opted for the selected antibodies because the signal strength is more continuous and better imitates the dilution or concentration gradient. Because little research has been done on the expression of GPR55 in breast cancer cell lines, we also confirmed the expression of GPR55 in the immortal T-47D breast cancer cell line based on the results.
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