In this experiment, we used insulin agarose column chromatography as the final step in insulin receptor purification. This method utilizes the high affinity binding capacity of insulin receptor for insulin tired (KD=10-9 mol/L). This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.

Operation method

Insulin affinity chromatography assay

Materials and Instruments

Partially purified insulin receptor NaCl Insulin agarose solution F solution G solution H
Disposable Small Plastic Columns CentriconTM Concentrator Silver Stain Kit

Move

MATERIALS AND EQUIPMENT

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

NaCl (5 mol/L)

Insulin agarose (~2 ml preloaded gel) (SigmaChemicalCo-)

Disposable mini-plastic columns (Bio-Rad Laboratories)

^CentriconTM concentrator ( Amicon, Inc.)

Silver Staining Kit (Bio-Rad Laboratories 161-0449)

Reagents

Solution F

Solution G

Solution H

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

Operating Procedures

1) Add 0.75 ml of 5 ml of 5mol/L NaCl to partially purified insulin receptor solution (~3 ml) from WGA-agarose column. Adjust the solution NaCl concentration to 1 mol/L.

2) Flush the insulin agarose with 40 ml of solution F to remove any uncoupled insulin that may interfere with the immobilization step.

3) Mix the mixture from step 1 with insulin agarose (0.5 ml insulin agarose per ml of receptor solution) and incubate at 4°C for 18 h. Incubate at 4°C for 10 minutes to remove any un-coupled insulin.

4) Allow the agarose to come to room temperature before injecting into a small disposable plastic column. small plastic columns. Wash the column with 15 ml of Solution F at room temperature.

5) Elute the insulin receptor with 10 ml of Solution G. Collect 1 ml of fraction. Collect 1 ml of the fraction in sections.

6) Identify the insulin receptor-containing fractions using the insulin binding assay described in Experiment 2 of this module (see P.212).

7) Combine the fractions with insulin binding activity and concentrate in a Centricon concentrator. This removes much of the water and detergent (the micelles of octyl beta-glucoside are about 8000 Da in size).

8) Mix with an equal volume of Solution H and add Triton X-100 back into the concentrated receptor solution. To obtain high affinity insulin binding activity and high insulin-stimulated autophosphorylation activity, it was necessary to replace octylβ-glucoside with Triton X-100 (Ridge et al., 1988).

9) In order to confirm that the product obtained was indeed the insulin receptor and its purity, the size of the isolated protein was determined by 7.5% SDS-polyacrylamide gel electrophoresis (see Appendix 5 ). Approximately 50ul of sample was added to each well, one of which was a molecular weight standard. After electrophoresis, the gel was silver stained using a silver stain plus kit.

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