ABCA1 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:500) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Protocol tips
-Rabbit (1:500) -Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Publication protocol

THP‐1 macrophages (0.8 × 106/well) were seeded in 6‐well plates and first treated with the indicated concentrations of piperine and pioglitazone for 24 h in FBS‐free RPMI‐1640 medium supplemented with 0.1% BSA and 10 μg/mL unesterified cholesterol. After incubation, cells were lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40; 1% protease inhibitor Complete (Roche); 1% PMSF; 0.5% Na3VO4; 0.5% NaF) for 30 min at 4°C. The lysed cells were collected and centrifuged at 16 060 g for 20 min to collect the supernatant. Protein concentration was assessed with Bradford assay using Roti®‐Quant from Carl Roth (#K015.1, Karlsruhe, Germany). Total protein (20 μg per sample) was loaded and separated via SDS‐PAGE. Expression levels of specific proteins were analyzed using antibodies against the indicated proteins and visualized with ECL reagent and a LAS‐3000 luminescent image analyzer (Fujifilm) with AIDA image analyzer 4.06 software (Raytest).

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Manufacturer protocol

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