Analytical experiments on glycosylation of insulin receptors

Summary

This experiment describes about the analysis of insulin receptor glycosylation. This experiment is from the Protein Purification and Identification Lab Guide by Houzhu Zhu.

Operation method

Analytical experiments on glycosylation of insulin receptors

Materials and Instruments

Purified insulin receptor Endoglycosidase F Endoglycosidase H Neuraminidase Glycerol Protease Inhibitor Reservoir Endoglycosidase F Buffer Endoglycosidase H Buffer Neuraminidase Buffer SDS-PAGE Sample Buffer
Gel electrophoresis device X-ray film and dark box

Move

Materials and equipment

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

Endoglycosidase F (N-glycanase; Boehringer Mannheim Corp. 903329 or Genzyme Corp.)

Endoglycosidases H (Boehringer Mannheim Corp. 100117 or Genzyme Corp. ENDO HI)

Neuraminidase (Boehringer Mannheim Corp.1080725 or Sigma Chemical Co. type X,N2133)

Gel electrophoresis device (BioRad Laboratories or Hoefer Scientific Instruments)

Glycerol (10%)

X-ray film and dark box

Reagents

Protease inhibitor reservoir solution (100X) (for glycosylation experiments)

Endoglycosidase F buffer (5x)

Endoglycosidase H buffer (10x)

Neuraminidase buffer (10x)

SDS-PAGE Sample Buffer (5x)

(For the recipe, see "Reagent Preparation", pp.234-240)

Shoving Procedures

1) Follow the procedure described in Experiment 5 of this unit (steps 1~5), and affinity label the insulin receptor of tube 5 purified by insulin-agarose column. Four identical tubes were prepared and labeled A, B, C, and D, respectively.
Note: The total volume of each tube is 62ul.

2) Set up the following reaction system:

Sample A (control)

a. Add 1ul of 100x Protease Inhibitor Reservoir Solution.

b. Add 17ul of _H2O.

Sample B (Endonuclease F Treatment)

a. Add 16ul of 5x Endoglycosidase F Buffer.

b. Add 2ul of Endoglycosidase F.

c. Add 1ul of 100x Protease Inhibitor Reservoir Solution.

Sample C (endoglycosidase H treatment)

a. Add 8ul 10x Endoglycosidase H Buffer.

b. Add 3ul of Endoglycosidase H.

c. Add 1ul of 100X Protease Inhibitor Reservoir Solution.

d. Add 6ul _H2O.

Sample D (neuraminidase treatment)

a. Add 8ul 10x Neuraminidase Buffer.

b. Add 3ul Neuraminidase.

c. Add lul 100x Protease Inhibitor Reservoir Solution.

d. Add 6ul of _H20.

3) Incubate the above samples at 37°C for 4 hrs.

4) Add 20ul of 5x SDS-PAGE sample buffer and stop the reaction.

5) Boil the samples for 5 min.

6) Add sample to a 7.5% SDS-polyacrylamide gel and electrophoresis at 170V until the dye front leaves the gel. (See Appendix 5 for further information on SDS-PAGE.)

7) Soak the gel in 10% glycerol for 10 min, then dry in a gel dryer.

8) Identify affinity-labeled insulin receptors by radioautography.

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