2016研究生受体第一次3学时.pptVIP

* Homogenate Precipitate Supernatant Precipitate Supernatant Precipitate Supernatant Nuclei, Unbroken cells Mitochondria, Membrane Microsomes 800-3000g,15min 10000-30000g, 15min 100000g, 1h Further separation of homogenate: differential centrifugation Cytosol * Common procedure 1. Total binding (radioactivity) – non-specific binding (radioactivity) 2. Conversion of radioactivity to mole 3. Design of suitable mathematical model 4. Curve fitting to yield required parameters and plot the curve Fundamental equation * Subtraction of Non-specific binding RT + LT ? TB RT + LT + unlabeled Ligand ?NSB LT LT Measured (TB) High affinity (SB) Low capacity) Low affinity (NSB) High capacity Optional * Conversion of cpm to mole cpm of the complex comes from ligand cpm÷(E) counting efficiency = dpm dpm÷(SA) specific activity (dpm/mol) of ligand = mole of ligand If ligand: receptor = 1, mole of ligand = mole of receptor If not, correct for the ratio Optional * * * Curve fitting by computer ? RT (Receptor amount) ? KL (Receptor affinity) RL2 – RL ?(KL+RT+LT) + LT?RT = 0 * Simplified Saturation Analysis: Single point method Asymptote * 2. Scatchard Plot: Straight line or curved line? * Curved Scatchard plot Multiple binding sites (ligand has different affinity) RT=0.005nmol/L KD=0.05nmol/L RT=0.01nmol/L KD=0.5nmol/L * Negative or positive co-operativity Negative cooperativity Positive cooperativity None * n=0.5, ratio of ligand to receptor = 1:2 n=1, ratio of ligand to receptor = 1:1 n=2, ratio of ligand to receptor = 2:1 2n1, or 0.5n1: negative or positive co-operativity or mixed subtypes with different affinity. RT RT-RL Log optional * 6. Dual sites saturation analysis RT=0.005nmol/L KD=0.05nmol/L RT=0.01nmol/L KD=0.5nmol/L * * 3. Competitive Binding Assay for comparing affinity Fixed radioligand, varying conc of unlabeled ligands