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Title of page!Dissociation ("off rate") experimentsA dissociation binding experiment measures the "off rate" for radioligand dissociating from the receptor. Initially ligand and receptor are allowed to bind, perhaps to equilibrium. At that point, you need to block further binding of radioligand to receptor so you can measure the rate of dissociation. There are several ways to do this:
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If the tissue is attached to a surface, you can remove the buffer containing radioligand and replace with fresh buffer without radioligand. |
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Spin the suspension and resuspend in fresh buffer. |
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Add a very high concentration of an unlabeled ligand. If this concentration is high enough, it will instantly bind to nearly all the unoccupied receptors and thus block binding of the radioligand. |
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Dilute the incubation by a large factor, perhaps a 20 to 100 fold dilution. This will reduce the concentration of radioligand by that factor. At such a low concentration, new binding of radioligand will be negligible. This method is only practical when you use a fairly low concentration of radioligand so its concentration after dilution is far below its Kd for binding. |
You then measure binding at various times after that to determine how rapidly the ligand falls off the receptors.
Each ligand-receptor complex dissociates at a random time, so the amount of specific binding follows an exponential dissociation (see Example model 2. Exponential).
| Variable |
Meaning |
Comment |
| X |
Time |
Usually expressed in units of seconds or minutes. |
| Y |
Total binding |
Usually expressed in units of cpm, fmol/mg, or sites/cell. |
| Span |
Difference between binding at time zero and plateau. |
Specific binding (same units as Y) |
| Plateau |
Binding that doesn't dissociate. |
Nonspecific binding (same units as Y). |
| K |
Dissociation rate constant often called koff. |
Expressed In units of inverse time (inverse of units of X-axis) |
| t1/2 |
Half-life |
0.69302/koff |
Analyzing dissociation data with Prism
To analyze dissociation binding data:
1. Enter the data with X equal to time after you initiated dissociation and Y equal to binding (usually total binding).
2. Perform nonlinear regression using the one phase exponential decay equation.
3. If you entered specific (rather than total) binding, make the variable PLATEAU a constant equal to zero. If you have entered total binding, leave the variable PLATEAU as a variable to fit.
Look at the nonlinear regression results. The variable K is the dissociation constant (koff or k-1) expressed in units of inverse time. If you entered the X values as minutes, koff is in units of min^-1. The results also show the half-life in units of time (minutes in this example).
Association binding experiments
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