Experiments for the determination of the number of insulin receptors on membranes
Experiments for the determination of the number of insulin receptors on membranes
Insulin binds to its receptor with high affinity (KD~109 mol/L), and this ability allows us to estimate the number of insulin receptors in even fairly crude preparations. This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.
Operation method
Experiments for the determination of the number of insulin receptors on membranes
Materials and Instruments
Binding buffer [125I] Insulin (receptor grade) Bovine serum albumin Porcine insulin Move Materials For more product details, please visit Aladdin Scientific website.
Bovine serum albumin (BSA, 1%)
Porcine insulin [17.5/umol/L (0.1 mg/ml) in 0.001mol/LHC1] (e.g., Sigma Chemical Co. 13505)
[125I ] insulin (receptor grade) (DuPontNEN NEX 196) (2200 uCi/mmol. 35.5 nmol/L)
Reagents
Binding Buffer (for two-site binding assay)
(for recipe, see Reagent Preparation PP.234-240)
Operating Procedures
1) Add 40ul of plasma membrane of rat liver (20 mg/ml) to each of the two microcentrifuge tubes, labeled #1 and #2.
2) Add 30ul of 1% BSA to each tube.
3) Add 140ul of Binding Buffer to each tube.
4) Add 60ul of 17.5 mg/ml of rat liver to each tube. Add 60ul of 17.5umol/L porcine insulin to tube #2 (to achieve a final concentration of 3.5umol/L), and 60ul of distilled water to tube #1.
5) Add 30ul of 5nmol/L [125I ] insulin to each tube (to achieve a final concentration of [125I ] insulin of 0.5nmol/L).
6) Incubate tubes on a rotary shaker at room temperature for 2 h or at 4 °C for 16 h. 7) Place tubes in a microcentrifuge.
7) Centrifuge the tubes in a microcentrifuge for 20 min. remove as much supernatant as possible.
Note: If the ligand has a high affinity for the receptor (e.g., KD=10-11 ), the precipitate can be washed to remove free ligand. However, if the ligand has only a low or moderate affinity for its receptor, washing may partially dissociate the ligand-receptor complex.
8) Measure the intensity of the radioactivity in the tube with a y-counter. Subtract the cpm value of tube #2 (non-specific binding count and background count) from the cpm value of tube #1 (total cpm value) to obtain the specific binding cpm value.
Calculation of insulin binding
The standard formula for hormone-receptor interactions is usually expressed as: 
where [H] is the concentration of free ligand, [R] is the concentration of free receptor, and [HR] is the concentration of the hormone-receptor complex. When equilibrium is reached, the dissociation constant is 
Thus, [H] = [H ]t - [HR] (where [H ]t is the total concentration of ligand) and [R ]t = [R] + [HR] (where [R ]t is the total concentration of receptor). Rearranging the equation, the KD is given as: 
For the two-site binding assay, the KD of the murine hepatic insulin receptor can be assumed to be 10-9 mol/L. The total concentration of receptor [R ]t can then be calculated from the measured [HR] and [H]. 
To determine the specific activity, it can be derived from either the amount of insulin bound, divided by the amount of protein in the sample, or by dividing the [R ]t by the concentration of protein in the test tube. This calculation is usually expressed as pml (insulin bound)/mg (protein).