PBL were separated on discontinuous Percoll gradients.
The isozymes of lipoxygenase (LOX) were separated on discontinuous polyacrylamide gels (stacking gel 4.5%, separating gel 10%) under non-denaturing and non-reducing conditions.
Nucleoproteins were separated on discontinuous SDS PAGE (9% gels), and then transferred by electroblotting to Immobilon membranes (Millipore, Bedford, MA, USA).
PNS collected from cultured INS-1 cells was separated on a discontinuous OptiPrep density gradient including five concentrations (8.8%, 13.2%, 17.6%, 23.4%, and 30%) (Fig. 1).
After washing, IEL and enterocytes were separated on a discontinuous density gradient (25 %, 40, and 75%).
The enzyme-digested cells and the cells in the supernatant were pooled and separated on a discontinuous Percoll density gradient (ρ = 1.01, 1.03, 1.05, 1.07 and 1.10) by centrifugation at 3,500 g for 20 min at RT. Light-density cells were collected from the second and third interfaces, which contain the majority of the thymic stromal cells.
Homogenized cell extracts were separated on a discontinuous density gradient (OptiPrep™).
Cells in the ascitic fluid were pelleted, resuspended in PBS, and separated on a discontinuous gradient consisting of (bottom to top) Lymphoprep (Axis-Shield, Oslo, Norway), Lymphoprep/Krebs HEPES Ring solution 3 1 and Lymphoprep/Krebs HEPES Ring solution 1 2 (Krebs HEPES Ring solution: 137 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.35 mM KH2PO4, 8 mM MgSO4, 1 mM HEPES pH 7.4).
The serum was concentrated prior to analysis and 150 µg of total serum protein was separated on a discontinuous 7 %/10 % acrylamide 1D SDS-PAGE gel, transferred to nitrocellulose and immunoblotted using proprietary polyclonal rabbit or monoclonal mouse antibodies raised against the cardiac-restricted C0C2 portion of cMyC (see below).
Equivalent amounts of total protein extract from each sample were electrophoretically separated on 10 15% discontinuous acrylamide gels, transferred to pure nitrocellulose membranes (PROTRAN BA85, Schleicher and Schuell, 0.45 µm), and the equal loading and transfer of the samples was monitored by Ponceau staining of the membranes.
After 19 h incubation in a humidified CO2/air incubator, the ring-enriched fraction was separated on and collected from a discontinuous 40/80/90% Percoll gradient, containing mannitol (6% wt/vol).
