MMP-9 Antibody (2C3): sc-21733

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The cell line samples were homogenized with ice-cold RIPA lysis buffer that was added with protease inhibitor (Complete EDTA-free, #10634200, Roche) and phosphatase inhibitor (PhosSTOP, #04906837001, Roche) and centrifuged at 20,000 x g for 30 minutes at 4°C. Supernatants were collected and kept in -80°C. Bradford method was used to quantify the protein concentration required for western blot sample loading (Pierce BCA Protein Assay Kit, #23225). All samples were dissolved in 1X LDS sample buffer and 1X reducing agent (Life Technologies) and heated for 5 minutes at 95°C. An equal concentration of protein was electrophoresed and separated in NuPage 4-12% Bis-Tris gel (Invitrogen, #NP0322) and transferred to a 0.2 um nitrocellulose membrane (#IB301032) using iBlot (Invitrogen). Blocking was carried out in 5% bovine serum albumin, 0.1% Tween 20 in PBS for non-specific binding for an hour at room temperature. The membranes were incubated overnight at 4°C with mouse monoclonal primary antibodies: OPN, LFMb-14 (1:200, sc-73631, Santa Cruz biotechnology); anti-flag (1:2000, F1804, Sigma), MMP9 2c3, (1:500, sc-21733, Santa Cruz Biotechnology), Anti-HS100A8 (1:500, MAB4570, R&D Systems,) and normalized with actin, C4 (1:5000, sc-47778, Santa Cruz Biotechnology). They were washed and incubated for 60 minutes at room temperature with secondary antibody, goat anti-mouse (1:5000, HRP, sc-2055, Santa Cruz Biotechnology). The membrane was visualized by chemiluminescent HRP substrate reagent (1:1) (Immobilon Western, WBKLS0500, Millipore) using ChemiDoc MP Imaging System (Bio Rad).

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Manufacturer protocol

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