This experiment describes the process of lectin affinity chromatography on solubilized receptors. This experiment was derived from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.

Operation method

Lectin affinity chromatography experiments on solubilized receptors

Materials and Instruments

Wheat Germ Agglutinin (WGA)-Agarose Dissolved Insulin Receptor Bovine Serum Albumin [125I] Insulin (Receptor Grade) Porcine Insulin Bovine Gamma-Globulin Polyethylene Glycol 6000 Solution D Solution E Binding Buffer
Disposable Small Plastic Columns

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MATERIALS AND EQUIPMENT

Wheat germ agglutinin (WGA)-agarose (~2.5 ml deposited microspheres) (Vector Laboratories, Inc. ALI023 or EY Laboratories, Inc. A2101)

Dissolved insulin receptor (obtained from Experiment 2)

Disposable miniplastic column (Bio-Rad Laboratories)

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/L HCl] (Sigma Chemical Co. I3505)

Bovine gamma-globulin ( 0.4%, in Alcoholic Solution B) (Miles Laboratories, Inc., 82-041-2)

Polyethylene Glycol 6000 (20% aqueous solution)

Reagents

Solution D

Solution E

Binding Buffer (for receptor solubilization)

(for recipe, see “Preparation of Reagents”, PP.234?240)

Operating Procedures

LectinAffinity Chromatography

1) Wash 2.5 ml of WGA-agarose with 25 ml of Solution D to remove uncoupled WGA.

2) Mix 10~15 mg of dissolved insulin receptor (~5 ml) with 2.5 ml of washed WGA-agarose and the mixture is gently shaken at 4°C for 16 h or at room temperature for 1 h.

3) Pour the agarose into a disposable mini-plastic column and rinse with 15 ml of solution D.

4) Elute insulin receptor with 15 ml of solution E. Collect 0.5-ml of solution E in portions. Collect the 0.5-ml fractions in sections and determine the protein concentration of each fraction by microprotdnassay. Combine the fractions containing the protein concentration peak; this typically yields approximately 3 ml of 0.3 mg/ml protein.
Note: The main protein peak is usually found in tubes 3-6, i.e., about 1/2 column volume, and is quite sharp. The reagent buffer and agarose for the sceptic column should be kept at the same temperature. Using reagents at different temperatures can create bubbles and cause inhomogeneity within the column, resulting in a loss of resolution of the eluted proteins.

Determination of insulin binding to partially purified receptors

1) Set up the following reaction system in microcentrifuge tubes:



The results from tube A give the total binding amount and the results from tube B give the non-specific binding amount. Specific binding = cpm value of tube A - cpm value of tube B.
Note: Receptor grade ^[125I ] insulin (NEX196) from DuPont NEN has a specific activity of 2200 uCi/nmoL. the insulin concentration is 35.5 nmol/L. For this assay, it is necessary to add [ 125I] to the [ 125I] insulin. _¹²⁵ I] insulin reservoir solution with buffer (50 mmol/L HEPES, 0.1% Triton X-100, 0.1% BSA, pH 7.4) to 5 nmol/L. For partially purified receptors, the final concentration of ^[125I ] insulin required for the binding assay should be 500 ppmol/L, which is comparable to 30 ul of 5 nmol/L solution in a 300-ul reaction system. 5 nmol/L solution in a 300-ul reaction system.

2) Incubate the above reaction system at 4°C for 16 h or at room temperature for 2 h.

3) Separate the receptor-bound insulin from the free insulin by precipitation. Add 75ul of 0.4% bovine gamma-globulin and incubate for 5 min at 4°C. Add 375420% polyethylene glycol 6000 and incubate for 10 min at 4°C.

4) The mixture was centrifuged in a microcentrifuge for 10 min and the precipitate was collected.

5) After removing as much of the supernatant as possible from the tubes, the sediment was counted to determine the radioactivity intensity.

Results

Typical results of purification of solubilized insulin receptor by WGA-agarose affinity chromatography are shown in Figure 4-5 and Table 5.

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