> Equilibrium Dialysis
Equilibrium dialysis measure the amount of a small molecule (ligand) bound to a protein at equilibrium. The method uses a chamber with two liquid compartments separated by a semi-permeable dialysis membrane. A solution of protein is placed in one chamber and a solution of the ligand is placed in the other chamber. The membrane separating the two chambers contains pores of suitable size to allow the ligand to pass easily between the two chambers but keeps the protein confined to one chamber.
As the ligand enters the chamber containing the protein, some of the protein binding to the protein while some remains free in solution. At equilibrium, the concentration of free ligand in both chambers is the same, but the chamber containing the protein also contains bound ligand. The amount of free ligand in each chamber can be determined by measuring the total ligand in the chamber that does not contain protein.
Measurement of the total ligand in the chamber containing the protein includes both free ligand and bound ligand. The amount of bound ligand can be determined by the difference in total ligand between the two chambers provided the volumes of solution in the two chambers are equal. If these values are determined for a series of ligand concentration, a binding curve can be constructed.
By fitting this binding curve to the equation:
Cbound=(Cfree*Lmax)/(Cfree+KD)
where,
Cbound is the difference between the amount of ligand in the chamber containing the protein and the chamber without protein.
Cfree is the amount of ligand in the chamber without protein
KD is the equilibrium binding constant for the interaction
Lmax is the maximum ligand binding capacity of the protein.
The value of KD for the interaction can be determined from the nonlinear least-squares fit of the binding data. Lmax in the above equation gives the amount of ligand bound at infinite ligand concentration. If the concentration of the protein is known accurately, the stoichiometry of the interaction can be calculated.
How to Order
- Consult with Proteomics staff concerning the detection method to be used and the specific sample requirements.
- Obtain a radioactively labeled form of the ligand, if needed
- Accurately determine the concentration of the protein and the specific radioactivity of the ligand.
- Print out the Equilibrium Dialysis Request Form as a word doc or pdf file./li>
- Submit the samples along with the completed request form
to the Proteomics Section in room D1011. Sufficient protein
and ligand should be submitted for approximately 10 dialysis
measurements. The concentration of the ligand should be high
enough that concentrations several times the KD can be achieved.
The binding curve will be calculated and the fitted binding curve will be provided via E-mail.
Quality Assurance
- Several concentrations of ligand are used and the resulting curve is analyzed by curve fitting programs.
- When possible, duplicate determinations will be done to ensure reproducibility.
For radioactive samples, the scintillation counter will be calibrated and appropriate quench corrections performed prior to data collection. Multiple counting cycles will be used to eliminate counting errors due to fluorescence or phosphorescence in the samples
Data Analysis/Troubleshooting
You will be notified by E-mail or telephone when the analysis is complete. At that time you should schedule a meeting with the Proteomics staff to discuss the results. During the meeting, the method of analysis and the controls used in the analysis will be described. The results of the experiment will be provided and any problems will be described. A hard copy of the final analyzed data and a written summary of the experiment will be provided. Suggestions of how to improve the data or further experiments which could provide more information will be discussed.
Instrumentation
Beckman LS6500 Liquid Scintillation Counter
Fees
Please follow this link to a general fees page.