All experiments have been performed in accordance with the U. S. Public Overall health Service Policy on Humane Care and Use of Laboratory Animals, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the ARVO Statement for the usage of Animals in Ophthalmic and Vision Study, and institutional, federal, and state recommendations relating to animal analysis. Groups of five 9 mice were subjected to optic nerve crush. Axons from the optic nerve had been crushed with fine forceps for ten sec, 2 mm pos terior to the globe, below direct visualization, inside an intact meningeal sheath. By performing the optic nerve crush two mm posterior towards the globe, the injury is distal for the entry from the ophthalmic artery into the optic nerve. As a result, care is taken to not disturb the retinal blood supply.
Optic nerve crush has been extensively made use of by several labora tories and is effectively documented inside the selleck inhibitor literature. In the desired instances eyes were enucleated and neural retina removed and frozen at 80 C. Controls were contralateral eyes that had not been injured in the similar animals in each group. Preparation of retinal extracts Retinal tissue was homogenized in 150lof lysis buffer NP 40, a number of protease inhibitor cocktail and 1 mM sodium orthovanadate. A motorized pes tle was used in three ? 20 sec bursts on ice. The mixture was centrifuged at 15,800 ? g for 20 min at 4 C. The superna tant was removed as well as the pellet re extracted with 50lof lysis buffer with mixing by up and down action of a pipette. The mixture was centrifuged once again as well as the super natants combined. Protein concentrations had been measured.
Method The initial screening for alterations in phosphorylated pro teins was completed applying affinity capture procedures coupled to mass spectrometry for protein identification. These meth ods incorporated anti phosphotyrosine beads for enrichment of tyrosine phosphorylated proteins followed by separa tion of your captured selleck chemical material applying one dimensional gel electrophoresis. We employed metal ion chelate chromatogra phy of phosphopeptides obtained from proteins that have been not captured by anti phospho tyrosine antibodies. We did these experiments at various points post injury to try to capture a broad spectrum of events in cellular signaling. Although the strategy employed was only semi quantitative, it lends itself to detection of changes in mul tiple phosphoproteins for each experimental time point.
Isolation of phosphoproteins peptides Tyrosine phosphorylated proteins have been isolated by immunocapture with anti phosphotyrosine antibody 4G10 conjugated to agarose beads. This antibody has been applied previously to character ize tyrosine phosphoryated proteins in stimulated cell sys tems and for quantitative phosphoprotein detection. Lysate containing 1 mg of protein was diluted 1,10 with lysis buffer without having NP 40 detergent and applied to 50lof a slurry of 4G10 conjugate.