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.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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