Mouse anti apoa1

Pre-heparin and post-heparin plasma samples were diluted 1:50 in 0.5-M Tris-HCl (pH 7). The diluted plasma samples, cell lysates, and concentrated media samples were mixed with Laemmli buffer and boiled for 5 min at 95 C. Proteins were size-separated by SDS-PAGE (10% acrylamide gel, 200 V, 45 min) and transferred onto a nitrocellulose membrane (400 mA, 60 min). Membranes were incubated overnight with primary antibodies, washed 2 times for 5 minutes with 0.2% tris-buffered saline Tween, incubated 1 hour with HRP-conjugated secondary antibodies and washed 3 times for 10 minutes with 0.2% tris-buffered saline Tween. After 5-minute incubation with chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA), bands were visualized by Chemidoc XRS System (Biorad, Hercules, CA) and Image Lab Software (Biorad).
The following antibodies were used: mouse anti-LPL (Sigma-Aldrich), rabbit anti-HTGL (Hepatic Triglyceride Lipase) (Sigma-Aldrich), mouse anti-APOA1 (AbD Serotec, Oxford, UK), mouse anti-V5 (Invitrogen), rabbit anti-Calnexin (Abcam), and mouse anti-Albumin (Sigma-Aldrich).

Post-heparin plasma samples were diluted 1:50 in 0.5-M Tris-HCl (pH 7). The diluted plasma samples, cell lysates, and concentrated media samples were mixed with Laemmli buffer 5X (SDS 10%, Tris HCl 62.5 mM pH 6.8, glycerol 50%, bromophenol blue 0.01% and β-mercaptoethanol 25%) and boiled for 5 min at 95 °C. Proteins were sizeseparated by SDS-PAGE (10% acrylamide gel, SDS 0.1%, 100 V, 90 min, using running buffer containing 0.1% SDS) and transferred onto a nitrocellulose membrane (400 mA, 60 min). Membranes were incubated for 1 h with primary antibodies, washed 2 times for 10 min with 0.2% tris-buffered saline containing 0.2% tween (TBST), incubated 1 h with HRP-conjugated secondary antibodies, then washed 3 times for 10 min with TBST. Membranes were incubated for 5 min with chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA). Bands were visualized by Chemidoc XRS System (Biorad, Hercules, CA) and quantified using Image Lab Software (Biorad). The following antibodies were used: mouse anti-LPL (Sigma-Aldrich) (1:1000), mouse anti-APOA1 (AbD Serotec, Oxford, UK) (1:500), mouse anti-V5 (Invitrogen) (1:5000), rabbit anti-Calnexin (Sigma-Aldrich) (1:2000) , mouse anti-Albumin (Sigma-Aldrich) (1:1000).