Immunological identification and isolation of phosphotyrosyl proteins in cultured cells

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6285756

Martensen, T M; Madoff, D H; Lane, M D

Affinity-purified sheep anti-phosphotyrosine antibodies were utilized to probe for phosphotyrosyl proteins from cultured cells. Western blots of cell extract proteins separated by SDS PAGE were incubated with anti-Tyr-P antibodies followed by peroxidase labeled anti-sheep antibodies or ( SVI)protein G to decorate the immune complexes. Normal, retrovirus transformed, and preadipocyte fibroblasts showed a variety of phosphotyrosyl proteins. Specific binding was indicated by the ability of Tyr-P but not Ser-P or Thr-P to inhibit the immunolocalization. Anti-phosphotyrosine antibodies covalently coupled to Sepharose were used to isolate phosphotyrosyl proteins from cultured cells. The binding capacity of the gel was determined by the quantity of unlabeled phosphotyrosyl protein needed to displace (TSP)labeled phosphotyrosyl protein binding. (TSP)labeled insulin receptor from 3T3-11 adipocytes could be immuno-adsorbed in high yield. Desorption was achieved in good yields under native conditions by incubation with Tyr-P. Treatment of (TSP)insulin receptor with SDS prior to incubation with immobilized antibody did not inhibit immuno-adsorption or -elution. This feature enabled antiphosphotyrosine antibodies to distinguish phosphotyrosyl proteins whose modified residues appear to be inaccessible for antibody binding in the native state.

• Research Organization: National Institutes of Health, Bethesda, MD
• OSTI ID: 6285756
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CULTURES
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
FAT CELLS
FIBROBLASTS
HORMONES
INSULIN
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RUMINANTS
SHEEP
SOMATIC CELLS
TRACER TECHNIQUES
VERTEBRATES