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Life Science > Proteomics > Recombinant Protein Expression > Protocols > Genetically Engineered Kinases
Proteomics & Protien Expression

Genetically Engineered Kinases
cshlp-small Sigma® has prepared product lists for commonly used protocols from Cold Spring Harbor Protocols.

View the entire protocol on BioSupplyNet.com.

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
Department of Microbiology and Cancer Center, University of Virginia Health System,
Charlottesville, VA 22908

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.   Protein Protein Interactions

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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.