Anti-LAMP1 antibody [1D4B] (ab25245)

Protocol tips

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	Primary Ab dilution-1:700 dilution

Protocol tips
Primary Ab dilution-1:700 dilution

Publication protocol

Cultured cardiomyocytes (35,000 cells cm–2), HL‐1 cells (3 × 105 in P6 wells) or heart tissues were lysed and subjected to SDS‐PAGE/western blotting as described previously (Lear et al. 2010). Briefly, cells were lysed with Triton X‐100 (1% in a buffer consisting of 50 mm Tris‐HCl, 150 mm NaCl, 5 mm EDTA, 1 mm phenylmethylsulphonylfluoride, 10 μg ml−1 leupeptin, 10 μg ml−1 aprotinin, 10 μg ml−1 trypsin inhibitor and 1 mm NaVO4). Samples were separated by 8%, 10% or 12% SDS‐PAGE (depending on the molecular weight of the protein) and transferred to polyvinyl difluoride membranes (GE Healthcare), which were incubated for 1 h in blocking solution (50 mm Tris‐HCl, 200 mm NaCl, 0.1% Tween 20 and 5% skimmed milk power) unless the manufacturer's instructions for the antibody indicated otherwise. The primary antibodies used were: TPC1 (dilution 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), TPC2 (dilution 1:200; Santa Cruz Biotechnology), Thr172‐phospho‐AMPK (dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK (dilution 1:1000; Cell Signaling Technology), Ser2448‐phospho‐mTOR (dilution 1:1000; Santa Cruz Biotechnology), mTOR (dilution 1:1000; Santa Cruz Biotechnology), LC3B (dilution 1:1000; Cell Signaling Technology), p62 (dilution 1:1000; Cell Signaling Technology), LAMP‐1 (dilution 1:700, Abcam, Cambridge, UK), Rab7 (dilution 1:1000, Cell Signaling Technology), GAPDH (dilution 1:1000; Sigma‐Aldrich) and β‐actin (dilution 1:1000; Thermo Fisher Scientific). Membranes were incubated for 1 h in horseradish‐peroxidase‐conjugated anti‐IgG secondary antibody (Santa Cruz Biotechnology) and visualized using an Immobilon HRP chemiluminescence detection system (Millipore, Billerica, MA, USA).

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Manufacturer protocol

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