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Life Science > Proteomics > Recombinant Protein Expression > Protocols > Genetically Engineered Kinases
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Proteomics & Protien Expression
Genetically Engineered Kinases | ||
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Sigma® has prepared product lists for commonly used protocols from Cold Spring Harbor Protocols. |
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Selected Protocol from "Protein-Protein Interactions", 2nd Edition, E. Golemis, P. Adam. Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber |
| ABSTRACT | ||
| Protein kinase cascades regulate many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effects on cellular processes. A recent development in this area takes advantage of the similarity in the ATP-binding domains of protein kinases. Conserved residues in the ATP-binding site contain large side chains that come into close contact with the N6 position of bound ATP. Mutation of one or more of these large residues to alanine or glycine generates a "pocket" in the ATP-binding site that allows the mutant kinase, but not the wild-type kinase or other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N6 position. Kinase reactions performed with a mutated kinase and radiolabeled ATP analogs in a cellular lysate allow specific labeling of direct substrates of the mutant kinase, which can later be identified by mass spectrometry or other means. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and the use of one such mutant to identify two novel substrates of ERK2 (Eblen et al. 2003). In this chapter, we discuss the generation and characterization of kinase mutants that utilize analogs of ATP, and describe the methods used to identify direct kinase substrates. These methods can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction. | 
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| Products Available for this Protocol | |||
| Protocol Material Description | Product # | Product Name | Add to Cart |
| Buffers, Solutions, and Reagents | |||
| Acetic acid | A9967 | Acetic acid | |
| Tris-buffered saline (TBS) | T6664 | Tris Buffered Saline | |
| NaCl | S3014 | Sodium chloride | |
| Water | W4502 | Water | |
| KCl | P9541 | Potassium chloride | |
| Tris-base | T6066 | Trizma® base | |
| Phosphate-buffered saline (PBS) | P5368 | Phosphate buffered saline | |
| KH2PO4 | 60353 | Potassium phosphate dibasic | |
| Sodium dodecyl sulfate (SDS) | L3771 | Sodium dodecyl sulfate | |
| Ponceau S solution | P7170 | Ponceau S solution | |
| Glycerol | G5516 | Glycerol for molecular biology | |
| Magnesium acetate | 63052 | Magnesium acetate solution, for molecular biology | |
| Dithiothreitol (DTT) | D9779 | DL-Dithiothreitol for molecular biology | |
| β-Mercaptoethanol | M3148 | 2-Mercaptoethanol for molecular biology | |
| Na2HPO4•2H2O | S9763 | Sodium phosphate dibasic | |
| 2x Laemmli sample buffer | S3401 | Sample Buffer, Laemmli 2× Concentrate | |
| DMEM | M3942 | MegaCell™ Dulbecco's Modified Eagle's Medium, liquid, sterile-filtered, cell culture tested | |
| Fetal Bovine Serum | F2442 | Fetal Bovine Serum sterile-filtered, cell culture tested, hybridoma tested | |
| NaF | S7920 | Sodium fluoride | |
| EGTA | E3889 | Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid | |
| EDTA | E7889 | Ethylenediaminetetraacetic acid disodium salt solution | |
| Triton X-100 | T8787 | Triton® X-100, for molecular biology | |
| Tetrasodium pyrophosphate | S6422 | Sodium pyrophosphate tetrabasic decahydrate | |
| Sodium vanadate | 590088 | Sodium metavanadate | |
| Aprotinin | A3428 | Aprotinin from bovine lung, lyophilized powder, 3-7 TIU/mg solid, cell culture tested | |
| Leupeptin | L9783 | Leupeptin hydrochloride | |
| Phenylmethylsulfonyl fluoride (PMSF), fresh | P7626 | Phenylmethanesulfonyl fluoride | |
| HEPES (pH 7.4) | 70216 | HEPES Sodium salt buffer solution | |
| ATP | A7699 | Adenosine 5'-triphosphate disodium salt | |
| BCA protein assay kit | BCA1 | Bicinchoninic Acid Kit for Protein Determination for 200-1000 µg/ml protein | |
| Plasmids | |||
| Expression plasmid of choice | Cloning & Expression | Expression plasmids | |
| Cells and Antibodies | |||
| COS-1 African green monkey kidney epithelial cells | 88031701 | COS-1 Cell Line from African green monkey kidney, SV40 transformed | |
| Antibodies to the epitope tag on the protein kinase of interest | Antibody Explorer | Antibodies | |
| A phospho-specific antibody to a known substrate of your kinase | Antibody Explorer | Antibodies | |
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| Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest. |