This experiment describes about the process of solubilization and activity determination of insulin receptor. This experiment is from the guide to protein purification and identification experiments, by Zhu Houzhu.

Operation method

Insulin receptor solubilization and activity assay experiments

Materials and Instruments

Purified murine liver plasma membrane Bovine serum albumin [125I] Insulin Porcine insulin Bovine gamma-globulin Polyethylene glycol 6000 Solution B Solution C Binding buffer
Ti 70 turntable and polypropylene tube with cap

Move

MATERIALS AND EQUIPMENT

Purified murine liver plasma membrane (from Experiment 1)

Ti 70 turn head and capped polypropylene centrifuge tube

Bovine serum albumin (BSA, 1%)

^[125I ] insulin (receptor grade, DuPont NEN NEX 196)

Porcine insulin [17.5 umol/L (0.1 mg/ml), in 0.001mol/LHCl] (Sigma Chemical Co. I3505)

Bovine gamma-globulin (0.4% in solution B) (Miles Laboratories Co. 82-041-2)

Polyethylene Glycol 6000 (20% aqueous)

Reagents

Solution B

Solution C

Binding buffer (for receptor dissolution)

(for recipe, see "Preparation of Reagents", pp. 234-240)

Procedures

Dissolve the receptor with TritonX-100. TritonX-100 Insulin Receptor Dissolution

Compare the efficiency of TritonX-100 with other descaling agents in dissolving insulin receptors from murine liver plasma membranes. The results are shown in Table 4-3.



1) Take 100-150 mg of pure murine liver plasma membrane, mix with 10 ml of Solution B, and then add 4 ml of Solution B. Incubate at 4°C for 30 min.

2) Suspend 200,000 g (Ti 70 head, 45,000 r/min) in a 30-minute centrifuge. 45000r/min) centrifuge for 30 min, collect the supernatant with a pasteurized pipette, taking care not to disturb the precipitate.
Note: TritonX-100-insoluble precipitate is often present and clings to the bottom of the tube.

Determination of Insulin Binding Activity to Solubilized Receptors

1) Set up the reaction system in a microcentrifuge tube as follows:



Total binding from tube A and non-specific binding from tube B. Specific binding = total binding.

Specific binding = total binding - non-specific binding.

Note: Receptor-pure ^[125I ] insulin (NEX 196) was purchased from DuPont NEN with a specific radioactivity of 2200 uCi/nmol. the insulin concentration was 35.5 nmol/L. In this assay, the ^[125I ] insulin reservoir must be diluted to 5 nmol/L with binding buffer. the final concentration of ^[125 I] insulin is required for receptor binding assays where the receptor is dissolved. The final concentration of [125I] insulin is 500 pmol/L, which corresponds to 30ul of 5 nmol/L solution in a 300-ul reaction system.

2) Incubate the reaction system at 44°C for 16 h, or at room temperature for 2 h.

3) Sedimentation, to separate receptor-bound insulin from free insulin. Add 75ul of solution B containing 0.4% bovine gamma-globulin and incubate for 5 min at 44°C. Add 375ul of 20% polyethylene glycol 6000 and incubate for 10 min at 44°C. Centrifuge the mixture in a microcentrifuge for 10 min and collect the precipitate. Remove as much supernatant as possible from each tube and count the intensity of the precipitate.

Note: Although the polyethylene glycol precipitation method is commonly used to determine the number of insulin receptor sites during purification, this method most often underestimates the absolute number of binding sites because only a portion of the insulin-receptor complex is precipitated (Finn et al., 1984). To determine the affinity of insulin for its receptor, several different concentrations of unlabeled insulin are added . Make the final concentration of insulinsof 500pmol/L to 200nmol/L as described in Experiment 1 of this unit.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/