Autophosphorylation experiments on insulin receptor stimulated by insulin
Autophosphorylation experiments on insulin receptor stimulated by insulin
In this experiment, we will examine the insulin-stimulated tyrosine kinase activity of the insulin receptor. The phosphorylated receptor will be detected with an anti-phosphotyrosine antibody (anti-phosphotyrosine antiboty), which can be isolated by SDS-PAGE. This experiment is from Protein Purification and Characterization Lab Guide by Houzhu Zhu.
Operation method
Autophosphorylation experiments on insulin receptor stimulated by insulin
Materials and Instruments
Purified murine hepatocyte plasma membrane Partially purified insulin receptor Purified insulin receptor Porcine insulin ATP Primary antibody Secondary antibody Autophosphorylation buffer Starting solution SDS-PAGE Sample buffer Protein blotting transfer solution Blocking buffer Tris-HCl NaCl Tween-20 Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Protein Gel Electrophoresis Device Whatman 3 MM Filter Paper Cellulose Nitrate Membrane Electrophoretic Transfer Tank ECLTM Immunoblotting Kit
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)
Porcine insulin [17,5 pmol/L (0.1 mg/ml), dissolved in 0.001 mol/L HCl] (e.g. Sigma Chemical Co. 13505) ATP (20 mmol/L; pH 7.4) Protein gel electrophoresis device (Bio-Rad Laboratories or Hoefer Scientific Instruments) What97 protein gel electrophoresis device (Bio-Rad Laboratories or Hoefer Scientific Instruments) Sigma Chemical Co. 13505)
ATP (20 mmol/L; pH 7.4)
Protein gel electrophoresis setup (Bio-Rad Laboratories or Hoefer Scientific Instruments)
Whatman 3 MM filter paper
Cellulose nitrate membrane (Schleicher and Schuell, Inc.)
Electrophoretic transfer tank (Bio-Rad Laboratories or Hoefer Scientific Instruments)
Primary antibody (anti-phosphotyrosine antibody) (monoclonal IgG 2bk; Upstate Biotechnology, Inc. 05-321)
Secondary antibody [horseradish Horseradish peroxidase (HRP)-labeled anti-mouse IgG; Bio Rad Laboratories]
ECLTM Immunoblotting Kit (Amersham Life Science, Inc.)
Reagents
Autophosphorylation Buffer (10x)
Starting Solution (10x)
SDS-PAGE Sample Buffer (5x)
Protein Blotting Transfers
Sealing Buffer
Tris-HCl (10 mmol/L; pH 7.4)
NaCl (150 mmol/L)
Tween-20 (0.1%) (TBST)
(For the recipe, see Preparation of reagents, pp.234-240)
Procedure
Autophosphorylation
1) Prepare the following mixture: 
2) Incubate the mixture at room temperature for 15 min.
3) Add 2ul 20 mmol/LATP and 4ul 3) Add 2ul 20 mmol/LATP and 4ul 10X starting solution to start the phosphorylation reaction. After mixing, incubate at room temperature for 15 min at
.
4) Terminate the reaction by adding 10ul of 5xSDS-PAGE sample buffer.
5) Boil the samples for 5 min.
6) Load the samples onto 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)
Transferring Proteins to Nitrocellulose Membranes
1) Place a fiber pad on one side of the gel holder, and a wet Whatman 3 MM filter paper on top of it.
2) Place the gel on top of the filter paper.
3) Wet a piece of nitrocellulose membrane with water and place it over the gel. Remove any air bubbles between the filter paper and the gel.
4) Place another moistened piece of Whatman 3 MM filter paper on top of the nitrocellulose membrane.
5) Lay another fiber pad on top of the nitrocellulose membrane to make a sandwich.
6) Close the gel holder.
7) Place the gel holder in the buffer tank with the nitrocellulose membrane facing the anode side of the tank. The sandwiches should be arranged as follows:
cathode (black)
fiber pad
3 MM filter paper
gel
cellulose nitrate membrane
3 MM filter paper
fiber pad
anode (red)
8) Fill the tank with protein blotting transfer solution.
9) Place in a cold room and electrophoretically transfer the blot for 2 h at 100 V.
Protein Blotting
1) Place the cellulose nitrate membrane in a closed buffer and incubate at 37°C for 1 h.
2) Place the cellulose nitrate membrane in a buffer with a sufficient amount of primary antibody (100 V). Keep the membrane moist and make sure the side of the nitrocellulose membrane facing the gel is up.
3) Shake the tray for 1-2 h at room temperature.
4) Wash the nitrocellulose membrane with 5 ml of TBST for 5 min. Repeat the procedure 4 times.
5) Add a sufficient amount of secondary antibody (5000x dilution of horseradish peroxidase-conjugated anti-mouse IgG) and incubate for 1 h at 37℃. enzyme-conjugated anti-mouse IgG), and keep the nitrocellulose membrane moist.
6) Incubate at room temperature for 1 h.
7) Wash the nitrocellulose membrane with 5 ml of TBST solution for 5 min, repeat 4 times.
8) Prepare chemiluminescent assay solution by taking Reagent 1 and Reagent 2 from ECL Protein Blotting Kit and mixing them together in equal quantities.
9) Pour the chemiluminescent solution onto the nitrocellulose membrane, react for 1 min.
10) Immediately drain off the excess reagent.
11) Place the nitrocellulose membrane in a plastic wrap.
12) Add the phosphorescentorientationdot.
Note: For this step, Polymark Natural Glow No. PM 501 works well and is available at craft stores.
13) Expose the protein blotting membrane for 5s, 1min or longer until the desired result is obtained.