Specific determination of tyrosine-phosphorylated proteins and peptides by differential iodination.
A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.
Schepky
A G
AG
Niedersächsisches Institut für Peptid-Forschung GmbH, Hannover, Germany.
Meinhardt
G
G
Austermann
S
S
Adermann
K
K
Schulz-Knappe
P
P
Forssmann
W G
WG
Hass
R
R
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007422:Intestines / Q000201:enzymology; D007455:Iodine / Q000737:chemistry / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000737:chemistry; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry*; D019000:Phosphotyrosine / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D011851:Radioactivity; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D019000:Phosphotyrosine; D007455:Iodine; D000469:Alkaline Phosphatase