Cross-linking experiments of the insulin receptor with [125I]insulin

Summary

This experiment describes about the cross-linking process of insulin receptor with [125I] insulin. This experiment is from the Experimental Guide for Protein Purification and Identification by Hauchu Zhu.

Operation method

Cross-linking experiments of the insulin receptor with [125I]insulin

Materials and Instruments

Purified murine hepatocyte plasma membrane Partially purified insulin receptor Purified insulin receptor [125I] insulin Porcine insulin Bis(succinimidoyl)octanedioic acid Dimethyl sulfoxide NH4Cl Glycerol SDS-PAGE Sample buffer
Protein Gel Electrophoresis Device Gel Dryer

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MATERIALS AND EQUIPMENT

Purified murine hepatocyte plasma membrane (see Experiment 1 of this unit)

Partially purified insulin receptor from WGA-agarose column chromatography (see Experiment 3 of this unit)

Purified insulin receptor from insulin agarose column chromatography (see Experiment 4 of this unit)

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

Porcine insulin (17.5 umol/L ) (e.g., Sigma Chemical Co. 13505)

Bis(succinimidoyl)octanedioic acid (Pierce Chemical Co. 21555)

Dimethylsulfoxide (DMSO)

_NH4Cl (1 mol/L)

Glycerol (10%; v/v)

Protein gel electrophoresis device (BioRad Laboratories or Hoefer Scientific Instruments)

Gel dryer (Bio-Rad Laboratories or Hoefer Scientific Instruments)

Reagents

SDS-PAGE Sample Buffer (5x)

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

Operating Procedures

1) Establish the following reaction system.



2) Incubate the reaction mixture at room temperature for 2 h.

3) Dissolve bis(succinimidoyl)octanedioic acid in DMSO to a final concentration of 10 mmol/L.

4) Add 6ul of 10 mmol/L bis(succinimidoyl)octanedioic acid to each tube, and incubate the reaction for 1 min at room temperature.

5) Terminate the reaction by adding 1ul of 1mol/L NH4Cl.

6) Add 15ul of 5xSDS-PAGE buffer. 7) Boil the sample.

7) Boil the sample for 5 min.

8) Add the sample to a 7.5% SDS-polyacrylamide gel. Electrophoresis was performed at 170V until the dye front ran out of the gel. (See Appendix 5 for more information on SDS-PAGE)

9) Incubate the gel in 10% glycerol and dry on a desiccator.
Note: Proteins can also be transferred to nitrocellulose membranes as described in Experiment 6 of this unit.

10) Identify affinity-labeled insulin receptors by radioautography.

Results

Typical results of cross-linking experiments are shown in Figure 4-6.

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