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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2013 Jun 15;1834(10):2045–2056. doi: 10.1016/j.bbapap.2013.06.003

Figure 4. Ligand binding on CB2-255 fusion protein in micelles.

Figure 4

A. Ligand binding on CB2 in detergent micelles. Saturation ligand binding was performed to determine the number of ligand binding sites (Bmax) and affinity (KD) of the interaction between CB2-255 fusion protein and CP-55,940 ligand. Aliquots of CB2-255 fusion protein from crude cell lysate, solubilized in buffer A supplemented with 10 μM CP-55,940 and immobilized onto 1D4-Sepharose resin (50 μL/sample) were washed with solutions of CP-55,940 (0-60 μM) in buffer A, supplemented with [3H]-CP-55,940 and analyzed for ligand binding as described in Material and Methods. Each point represents an average of duplicate measurements. Results of a representative experiment (out of three) are presented.

B. Thermoinactivation of CB2 protein in micelles. The CB2-255 fusion protein from crude lysate in buffer A supplemented with 10 μM CP-55,940 was immobilized on 1D4-Sepharose resin (50 μL/sample) and subjected to a temperature increase from 4 °C to 84 °C at a rate of 1 °C/min. Functional activity of CB2 was analyzed by measuring ligand binding upon addition of [3H] CP-55,940 as described in Materials and Methods. Measurements were performed in duplicate; a representative experiment is shown. Squares- buffer without CHS, circles- buffer with CHS. Results of a representative experiment (three total) are presented.