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Table of contents
Intro to regression
Nonlinear regression
Curve fitting with Prism
Interpreting the results
Comparing two curves
Distributions of best-fit values
Radioligand binding
Saturation binding


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Introduction
Nonspecific binding
Fitting specific binding
Fitting  total binding
Analysis checklist
Scatchard plots
Ligand depletion
Competitive binding
Kinetics of binding
Dose-response curves
Enzyme kinetics
Standard curves
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Checklist for saturation binding

When evaluating results of saturation binding analyses, ask yourself these questions:

Question
Comment
Did only a small fraction of the radioligand bind? The analysis assumes that the free concentration is almost identical to the concentration you added. You can test this by comparing the total cpm that bound to the total cpm added to the tube. If more than 10% of the ligand bound (at any ligand concentration) then the standard analysis won't work. Either change the experimental protocol (increase the volume) or use a method that accounts for depletion of radioligand -- see Analyzing saturation binding with ligand depletion.
Did the binding equilibrate? The tubes with the lowest concentration of radioligand take the longest to equilibrate. So test equilibration time with a low concentration of radioligand.
Did you use high enough concentrations of radioligand? Calculate the ratio of the highest radioligand concentration you used divided by the Kd reported by the program (both in nM or pM). The highest concentration should be at least 10 times the Kd, so that occupancy exceeds 90%.
Is the Bmax reasonable? Typical values for Bmax are 10-1000 fmol binding sites per milligram of membrane protein, 100-10000 sites per cell or 1 receptor per square micron of membrane. If you use cells transfected with receptor genes, then the Bmax may be many times larger than these values.
Is the Kd reasonable? Typical values for Kd of useful radioligands range between 10 pM and 10 nM. If the Kd is much lower than 10 pM, the dissociation rate is probably very slow and it will be difficult to achieve equilibrium. If the Kd is much higher than 10 nM, the dissociation rate will probably be fast, and you may be losing binding sites during separation of bound ligand from free radioligand.
Are the standard errors too large? Are the confidence intervals too wide? Divide the SE of the Bmax by the Bmax, and divide the SE of the Kd by the Kd. If either ratio is much larger than about 20%, look further to try to find out why.
Is the nonspecific binding too high? Divide the nonspecific binding at the highest concentration of radioligand by the total binding at the highest concentration. Nonspecific binding should usually be less than 50% of the total binding.


Scatchard plots                                                                                                                                                                                                                                                                                                            

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