Materials Reagents

The protocol describes the measurement of Akt/PKB kinase activity in cultured 3T3-L1 adipocytes, a model cell line used for studying insulin-stimulated signal transduction and metabolism. The following reagents are used for the maintenance and differentiation of the 3T3-L1 line.

Fig. 2. Example kinase assay done using three different Akt/PKB substrates. Assays were performed as described in the text using 3T3-L1 preadipocytes treated with or without insulin as indicated. (A) Assay was performed using Bad as a substrate, and detection was performed using the phospho-Bad antibody. The top band is the full-length recombinant 31-kDa protein, and the lower band is degradation product. (B) Recombinant GSK3ß was used as a substrate, and the amount of phosphate transferred into GSK3ß from [y-32P]ATP was determined by autoradiogram. (C) Histone H2B was used as a substrate, and the amount of phosphate transferred into H2B from [y-32P]ATP was determined by autoradiogram.

Fig. 2. Example kinase assay done using three different Akt/PKB substrates. Assays were performed as described in the text using 3T3-L1 preadipocytes treated with or without insulin as indicated. (A) Assay was performed using Bad as a substrate, and detection was performed using the phospho-Bad antibody. The top band is the full-length recombinant 31-kDa protein, and the lower band is degradation product. (B) Recombinant GSK3ß was used as a substrate, and the amount of phosphate transferred into GSK3ß from [y-32P]ATP was determined by autoradiogram. (C) Histone H2B was used as a substrate, and the amount of phosphate transferred into H2B from [y-32P]ATP was determined by autoradiogram.

1. Dulbecco's modified Eagle's-H21 medium, Leibovitz L-15 medium, fetal bovine serum, and calf serum can be obtained from Invitrogen (Carlsbad, CA).

2. Dexamethasone can be prepared as a 4 mg/mL stock solution in ethanol; this is stable for several months if stored at -20°C.

3. Isobutylmethylxanthine can be prepared as a 5 mM stock solution in isotonic saline; this will require boiling to dissolve, but can be filter-sterilized and then stored at -20°C until use.

4. Insulin solution: A stock insulin solution can be prepared by diluting enough insulin in 0.005 N HCl to make an approx 300 iM stock solution. The final insulin concentration should be determined by ultraviolet (UV) absorbance. This solution may be stored for several months at 4°C.

5. Lysis buffer: 20 mMTris-HCl (pH 7.2), 150 mMsodium chloride, 10% glycerol, 1% igepal, 10 mM sodium fluoride, 30 mM sodium pyrophosphate, and 1 mM EDTA. This solution can be stored for 6 mo when filter-sterilized and kept at 4°C. The following protease and phosphatase inhibitors must be added fresh: 1 mM Na3VO4, 1 mM polymethylsufonyl fluoride (PMSF), 10 ig/mL leupeptin, and 2 ig/mL apro-tinin (see Note 1).

6. Protein assays: Protein concentrations should be determined using a method that is unaffected by detergents found in the lysis buffer. We use the bicinchoninic acid (BCA) protein assay kit from Pierce Chemical Company (Rockford, IL).

7. Antibodies: Isoform-specific anti-Akt antibodies generated against a regulatory domain near the carboxyl terminus are available from Upstate Biotechnology, Inc. (Lake Placid, NY). New England Biolabs also sells antibodies recognizing different Akt/PKB isoforms, as well as antibodies recognizing the phosphorylated form of the enzyme (4,6). Agarose-conjugated protein-A and/or secondary antibodies used to extract the antibody bound Akt from the lysate are available from Santa Cruz Biotechnology (Santa Cruz, CA) (see Note 2).

8. Substrate: Histone H2B can be purchased from Roche Applied Science (Indianapolis, IN) and Bad can be purchased from Upstate Biotechnology, Inc. GSK3b can be produced as a recombinant fusion protein with glutathione-S-transferase using standard techniques. Substrates can be stored in 50% glycerol at -20°C. If using histone H2B, include 25 ig per reaction. For GST-GSK3P or Bad, use 15 ig or 3 ^g per reaction, respectively.

9. Kinase reaction solutions: Prepare a 20X kinase buffer (pH 7.2) containing 400 mM HEPES and 100 mM MgCl2. This solution can be stored for several months at 20°C. Prepare the following kinase mix the day of the experiment: 1 mM dithiothreitol (DTT), 10 iM MgATP, 200 iM EGTA, 2 ig protein kinase inhibitor, and the appropriate amount of substrate. Ten microliters of this kinase mix containing 5 iCi of 6000 Ci/mMol [y32P] ATP is added to each reaction (see Note 3). The DTT and EGTA can be kept as 100X stock solutions, and the MgATP can be frozen as a 100X stock. All reagents can be obtained from Sigma (St. Louis, MO).

10. Sample solubilization buffer (SSB) (2X stock): 125 mM Tris-HCl (pH 6.8), 5.5% sodium dodecyl sulfate (SDS), 20% glycerol, 10% b-mercaptoethanol, and 0.006% bromophenol blue.

Delicious Diabetic Recipes

Delicious Diabetic Recipes

This brilliant guide will teach you how to cook all those delicious recipes for people who have diabetes.

Get My Free Ebook


Post a comment