Pull-down and identification of the ligand

Venae cavae were dissected from wild-type mice and fat and connective tissue were removed.  The endothelial tissue was placed in lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% IGEPALCA-630, 5 mM Na₃VO₄,  0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 0.5 µg/ml pepstatin) and homogenized with a PowerGen homogenizer (Fisher Scientific, Loughborough). Lysates were centrifuged at 13,000× g for 10 minutes at 4°C. Supernatants were collected and re-centrifuged under the same conditions.  Protein lysate (8 mg in 4.5 ml buffer) was precleared with Protein G sepharose (PGS, 50% slurry; 100 µl per mg protein,) and human IgG-Fc fragment (10 µg per mg protein) by agitation for 1 h at 4°C. The PGS beads were pelleted and the supernatant was then split into two samples which received either 50 µg mG6b-B-Fc or 50 µg human IgG-Fc fragment (negative control).  After 1.5 h 200 µl PGS was added and samples were agitated for another 1.5 h at 4°C.  Finally, PGS was washed three times in lysis buffer w/o protease inhibitors (by pelleting beads at 9,000 rpm for 20 sec, at 4°C and each time discarding the supernatant) and bound proteins were eluted by boiling the PGS pellet for 5 min at 105°C in 40 µl 2x SDS sample buffer.  The samples were then resolved on a NuPage 4-12% Bis-Tris-Gradient Gel (Invitrogen), alongside with 40 µg of mG6b-B-Fc (additional negative control) and stained with Colloidal Coomassie (National Diagnostics) according to the manufacturers protocol.  Bands appearing in the mG6b-B-Fc pulldown, but not in the negative controls, were excised and subjected to mass spectrometry analysis (Orbitrap, Thermo Fisher Scientific, Paisley, UK).  Corresponding areas from the control pulldown were cut and analyzed in parallel to account for background signals. 

2× SDS Sample buffer:

Add a trace of Brilliant Blue R dye.

Bring volume up to 10 ml with deionized water.

Can be stored at room temperature.