Experiments for the determination of the affinity of the insulin receptor for insulin

Summary

This experiment describes the method of determining the affinity of insulin receptor for insulin. This experiment is from the Experimental Guide for Protein Purification and Characterization by Houzhu Zhu.

Operation method

Experiments for the determination of the affinity of the insulin receptor for insulin

Materials and Instruments

Bovine blood albumin Porcine insulin [125I]insulin (receptor grade) binding buffer

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Materials

^[125I ]insulin (receptor grade) (DuPontNENNEX196)

Porcine insulin (e.g., Sigma Chemical Co, I3505)

Bovine blood albumin (BSA; 1%)

Reagents

Binding buffer (for cold ligand substitution experiments)

(for the recipe, see Reagent Preparation PP.234~240)

Procedures

1) The following reaction system was set up in microcentrifuge tubes: 2) Each tube was incubated on a shaker for 2 h at room temperature to remove the supernatant. 3) Each tube was centrifuged in a microcentrifuge for 20 min to remove the supernatant. The following reaction system was set up in microcentrifuge tubes:



2) The tubes were incubated on a shaker for 2 h at room temperature.

3) The tubes were centrifuged in a microcentrifuge for 20 min, and the supernatant was removed.

4) The radioactivity in the tubes was measured with a γ;-counter. Subtract the cpm value of tube 7 (non-specific cpm value and background cpm value) from the cpm values of tubes 1 to 6, respectively, to get the specifically bound cpm value.

5)Divide the specifically bound cpm value of each tube by the total cpm value to get the share of that portion of insulin bound to the membrane. This value is multiplied by the insulin concentration in each tube to obtain the concentration of the ligand-receptor complex ([HR], also known as [B], which represents bound ligand).

6) Subtracting [HR] from the insulin concentration in the tubes yields the concentration of the free ligand ([H], also known as [F], which represents free ligand).

7) At this point, the results obtained can be analyzed by a number of methods. It would be better to use a computer program to substitute the data directly into the following equation:



The data can also be analyzed by plotting [HR] against log([H]). This plot should give an S-curve. Thus, the total number of receptors and the _KD value can be derived from the maximum value of [HR] and the inflection point of the curve, respectively (Klotz, 1982). The third method is to analyze the data using a Scatchard plot, which is the plot made by . If there is only one type of binding site, the Scatchard plot is a straight line. In order to make a Scalchard diagram, the [HR]/[H] value corresponding to each [H] is determined, and then the [HR]/[H] value is plotted against [HR]. The slope of the line is _-1/KD, and the intercept of the horizontal coordinate is the total number of receptors ([R _]t ). If there is more than one type of binding site with different affinities, the Skatchelder plot will be a curve rather than a straight line.

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