Mutant Kinase ATP Analog Pair

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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.		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

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