Anti-β2-Microglobulin antibody produced in rabbit

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	-Rabbit (1:500)

Protocol tips
-Rabbit (1:500)

Publication protocol

Endothelial cells (3×106 cells) cultured in 10-cm dishes were serum starved for 4 h and treated with 1 µM S1P and 40 ng/ml VEGF and bFGF for 30 min. Cells were placed on ice, washed twice with 10-ml cold PBS with cations (1.5 mM Mg2+ and Ca2+), lysed in 700 µl cold lysis buffer containing 0.5% NP-40 in PBS with cations, 1× protease inhibitor cocktail (Roche, Mannheim, Baden-Württemberg, Germany) and 100× HALT phosphatase inhibitor (Thermo Scientific, Ashville, NC), and incubated for 10 min on ice with occasional mixing. Lysates were centrifuged at 16,000 g for 15 min at 4°C. Supernatants were collected and precleared with protein-G-conjugated magnetic beads (Invitrogen) (5 µl) for 1 h at 4°C with agitation. Supernatants were incubated with 2 µg antisera directed against MT1-MMP (ab38971, Abcam) or FAK (ab40794, Abcam) or species-specific IgG control for 18 h at 4°C with agitation. Protein-G-conjugated magnetic beads (10 µl) were added for 2 h at 4°C. Beads were washed five times with 1 ml lysis buffer without protease inhibitors, eluted in 1% SDS and analyzed by performing western blotting.

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