Fp 2020 plus

The amino acid compositions of four BRPFs were identified using high-performance liquid chromatography (HPLC),
following the AOAC method [18 ]. Samples were hydrolysed with 6 N HCl at 110 °C
for 24 h and derivatised using the AccQ-Fluor reagent. All sample solutions were then analysed using HPLC (Waters Alliance
2695, USA) coupled to a fluorescence detector (JASCO FP2020 Plus, Japan), with the excitation and emission wavelengths set at
250 and 395 nm, respectively. A AccQ Tag column (3.9mm×150 mm, 4 mm, Waters, USA) was used and eluted at a flow rate of
1 mL/min in gradient mode using a mixture of eluent A (sodium acetate buffer pH 4.90) and eluent B (60% acetonitrile). Amino
acid compositions were identified and quantified by comparison with a standard mixture of 17 amino acids (Sigma-Aldrich, USA)
and were shown as g of amino acid per 100 g of protein. Under strong acid hydrolysis condition, the recovery of each amino
acid located between 80% and 110% regardless of whether they were partially destroyed. The determination of cysteine and
methionine content, the samples were oxidized with performic acid before hydrolysis with 6N HCl [19 ] and tryptophan was determined by alkaline hydrolysis at 105 °C for 24 h with 4M NaOH
[20 ].

Purified CB1-eGFP (1.25 M excess) was incubated with G_i (that has been incubated with 1 % L-MNG for 1 hour), in the presence of the tested ligands at room temperature for 1 hour, after which apyrase was added and further incubated on ice for 1 hour. The sample was applied to a Superdex 200 10/300 column (equilibrated in 20mM HEPES pH 7.5, 100mM NaCl, 0.01% L-MNG/0.001% CHS) in-line with a Jasco FP 2020 Plus fluorescence detector set to an excitation of 480 nm and an emission of 512 nm. Complex formation and complex stability was monitored for each ligand by analysing the extent of free receptor and complex peaks.