Non immune mouse igg

Human platelets (5 × 10⁹ cells/10 ml) were washed with cold PBS and lysed in buffer containing 20 mM HEPES pH 7.40, 150 mM NaCl, 5 mM EDTA, 1% Brij 99, and protease inhibitors (HALTS, Pierce). After 30 min at 4 °C, insoluble material was removed by centrifugation at 16,000 ×g (15 min, 4 °C). The platelet lysates were pre-cleared by adding 20 μg of non-immune mouse IgG (Sigma) and 200 μl of Protein G-Sepharose (Amersham Pharmacia Biotech) and rocking gently at 4 °C for 60 min. Immunoprecipitation was performed by combining 20 μg of the anti-TSP-1 mouse monoclonal MA-IV and 200 μl of Protein G-Sepharose. The samples were incubated for 16 h at 4 °C with gentle rocking. Immune complexes were collected by centrifugation, washed four times in lysis buffer, and separated by SDS-PAGE in the presence of a reducing agent. Coomassie Blue stained bands were subjected to in-gel reduction, carboxyamidomethylation and tryptic digestion (Promega). Multiple peptide sequences were determined in a single run by microcapillary reverse-phase chromatography which was directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer equipped with a custom nano-electrospray source. The Harvard Microchemistry Facility completed this analysis on a fee-for-service basis (Miao et al., 2001b (link)).

The use of human tissue paraffin embedded samples was approved according to the Institutional Review Board Administration. The “BC20812 and “BRC711” breast cancer tissue arrays with pathology grading were obtained from US Biomax, Inc. (Rockville, MD). Grade II and III tumors were analyzed in this study. Tissue arrays were deparaffinized with xylene and hydrated with graded ethanol solutions. Antigen retrieval was performed using heated citrate buffer, reduced pH (Bethyl Laboratories, Montgomery, TX). The arrays were blocked with 2 % bovine serum albumin in PBS, and immunostained overnight at 4°C^{5 (link)}. Primary antibodies were mouse anti-matriptase (1:200), goat anti-c-Met (1:200) and rabbit anti-phospho-cMet (1:200) (R&D Systems, Minneapolis, MN), rabbit anti-matriptase (1:100) (Calbiochem/EMD Millipore, San Diego, CA), rabbit anti c-Met (1:30)(Leica Microsystems Inc., Buffalo Grove, IL) and rabbit anti-phospho-cMet (1:200) (Abcam, Cambridge, MA). As negative controls, non-immune mouse IgG, rabbit IgG or goat IgG were used (Sigma, St. Louis, MO) (adjusted to same final concentration as specific primary antibodies).