• Product Directory

    Custom Product

  • Services Offered

    Custom Capabilities
Decrease Font Size Increase Font Size Email this page to a friend Printer Friendly Page
Life Science > Proteomics > Recombinant Protein Expression > Protocols > Mutant Kinase ATP Analog Pair
Proteomics & Protien Expression

Mutant Kinase ATP Analog Pair
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.

Identification of a Mutant Kinase/ATP Analog Pair

Margret B. Einarson and Elena N. Pugacheva
Foxx Chase Cancer Center, Philadelphia, PA 19111

Jason R. Orlinck
Brigham & Women’s Hospital, Boston, MA 02115

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.   Protein Protein Interactions

Buy this book, Z719145

Products Available for this Protocol
Protocol Material Description Product #  Product Name Add to Cart
Buffers & Solutions      
50 mm Tris-base (pH 7.4) T6066 Trizma® base, cell culture tested, meets EP & USP testing specifications, Biotechnology Performance Certified
   
150 mm NaCl S3014 Sodium chloride for molecular biology
   
10% glycerol G5516 Glycerol
   
1% Triton X-100 X100 Triton® X-100
   
0.5 mm EDTA E6758 Ethylenediaminetetraacetic acid
   
0.5 mm EGTA E3889 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
   
50 mm NaF S7920 Sodium fluoride
   
40 mm β-glycerophosphate G1150 Glycerol 2-phosphate disodium salt hydrate
   
5 mm tetrasodium pyrophosphate S6422 Sodium pyrophosphate tetrabasic decahydrate
   
0.1 mm sodium vanadate 72060 Sodium metavanadate
   
10 µg/ml aprotinin A3428 Aprotinin from bovine lung
   
5 µg/ml leupeptin L8511 Leupeptin hemisulfate salt
   
2 mm phenylmethylsulfonyl fluoride (PMSF), fresh P7626 Phenylmethanesulfonyl fluoride
   
Acetic acid (1%) 338826 Acetic acid
   
250 mm HEPES (pH 7.4) H4034 HEPES
   
100 mm magnesium acetate 63052 Magnesium acetate solution
   
10 mm dithiothreitol (DTT) D9779 DL-Dithiothreitol
   
10 µm ATP A6559 Adenosine 5'-triphosphate disodium salt solution
   
PBS P5368 Phosphate buffered saline
   
25.6 g of Na2HPO4 - 7H2O S3264 Sodium phosphate dibasic
   
80 g of NaCl S3014 Sodium chloride
   
2 g of KCl P9541 Phenylmethanesulfonyl fluoride
   
2 g of KH2PO4 P9791 Potassium phosphate monobasic
   
Ponceau S solution 78376 Ponceau S
   
2x Laemmli sample buffer S3401 Sample Buffer, Laemmli 2× Concentrate
   
100 mm Tris (pH 6.8) T6066 Trizma® base, cell culture tested, meets EP & USP testing specifications, Biotechnology Performance Certified
   
2% SDS L4390 Sodium dodecyl sulfate
   
20% glycerol G5516 Glycerol
   
4% β-mercaptoethanol (added fresh) M3148 2-Mercaptoethanol
   
Dulbecco's modified Eagle medium (DMEM) Cell Culture Dulbecco's modified Eagle medium (DMEM)  
Fetal bovine serum (FBS) F2442 Fetal Bovine Serum
   
Cells & Antibodies      
COS-1 African green monkey kidney epithelial cells (ATCC) 88031701 COS-1 Cell Line from African green monkey kidney
   
Antibodies to the epitope tag on the protein kinase of interest Antibody Explorer Antibody Explorer  
A phospho-specific antibody to a known substrate of your kinase Antibody Explorer Antibody Explorer  
Additional Reagents      
BCA protein assay kit BCA1 Bicinchoninic Acid Kit for Protein Determination
   
Nitrocellulose membrane N7892 Nitrocellulose membranes for blotting
   
Vacuum flask Z290483 Vacuum filtration assembly flasks
   

back to top
View all available product lists for Cold Spring Harbor Laboratory Protocols
 
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.