Phosphotyrosine in proteins: chemical and immunological identification and isolation of phosphotyrosyl proteins

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5334329

Martensen, T M

Proteins which contain phosphotyrosine (Tyr-P) residues can be identified and purified by utilizing the chemical and immunological properties of the Tyr-P moiety. The stability of Tyr-P to basic conditions which cleave most ser/thr phosphoryl bonds enabled the analysis of cellular phosphoproteins after Na dodecyl SO/sub 4/ gel electrophoresis. Analyses were simplified using a nylon electroblot of the gel which is stable to 1 hr incubation in 1 N NaOH at 65/sup 0/C. Autoradiograms of nylon blots before and after base treatment were performed on /sup 32/P-labeled phosphoproteins of normal and retrovirus transformed fibroblasts. Conditions for optimal labeling of cellular proteins were studied by protein tyrosine kinase activity measurements using poly glu/sub 4/tyr. Differences in base resistant phosphoprotein patterns could be seen for each transformed cell line. Confirmation that certain base resistant phosphoproteins contained Tyr-P was achieved by immunostaining with affinity-purified sheep anti-Tyr-P antibodies (Ab). These Ab served as useful reagents to study Tyr-P proteins since they inhibit their dephosphorylation by phosphatases, and precipitate them in the presence of rabbit anti-sheep IgG Ab. Tyr-P protein was immobilized when mixed with anti-Tyr-P Ab and incubated with immobilized protein A sepharose rabbit anti-sheep IgG Ab complex. Selective elution of the Tyr-P protein was achieved with free Tyr-P.

• Research Organization: NHLBI, NIH, Bethesda, MD
• OSTI ID: 5334329
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
HYDROXY ACIDS
ISOTOPES
LABELLING
LIGHT NUCLEI
MAMMALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
RABBITS
RADIOISOTOPES
RUMINANTS
SHEEP
TRANSFERASES
TYROSINE
VERTEBRATES