CelLytic™ M

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	- Cells are lysed using CelLytic MT mammalian tissue lysis/extraction reagent containing 1% complete protease inhibitor mixture according to the manufacturer’s protocol.

Protocol tips
- Cells are lysed using CelLytic MT mammalian tissue lysis/extraction reagent containing 1% complete protease inhibitor mixture according to the manufacturer’s protocol.

Publication protocol

Caco-2 cells were lysed using CelLytic MT mammalian tissue lysis/extraction reagent (Sigma-Aldrich Co., MO, USA) containing 1% complete protease inhibitor mixture (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, the sample was homogenized and centrifuged at 5,000 g at 4 °C for 10 min. Supernatant was designated as whole cell lysate. Half of the supernatant was recentrifuged at 100,000 g for 2 h. The supernatant fraction from this step was designated as cytosolic fraction while the pellet was resuspended by the same buffer and used as membrane fraction. Samples were stored at -80 oC prior to use. For western blot analysis, protein concentration was determined, resolved in 4× Laemmli solution, electrophoresed on 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (GE Healthcare, WI, USA). Non-specific binding was eliminated by blocking with 5% (w/v) non-fat dry milk in 0.05% Tween® 20 in tris-buffered saline for 1 h. Polyclonal anti-rabbit NPC1L1, monoclonal anti-mouse alkaline phosphatase, or antimouse β-actin antibody were incubated at 4 °C overnight. Polyvinylidene difluoride membrane was washed with tris-buffered saline and incubated with horseradish peroxidaseconjugated immunopure secondary goat antirabbit or antimouse IgG (Merck, Darmstadt, Germany) for 1 h. The target protein was detected using Super Signal West Pico Chemiluminescent substrate (GE Healthcare, WI, USA) and quantitatively analyzed with the Image J program from the Research Services Branch of the National Institute of Mental Health (Bethesda, MD, USA).

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for CelLytic™ M below.

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