CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in RIPA buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 0.1% sodium dodecyl sulfate.	- Dilute primary Ab at 1 : 1000 and incubate membranes overnight.

Upstream tips
- Lyse cells in RIPA buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 0.1% sodium dodecyl sulfate.
Protocol tips
- Dilute primary Ab at 1 : 1000 and incubate membranes overnight.

Publication protocol

Cardiomyocytes were lysed in RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 0.1% sodium dodecyl sulfate), and 35 μg of proteins was resolved by SDS-PAGE, transferred to a PVDF membrane (by the Trans-Blot Turbo Transfer System, Bio-Rad), and immunoblotted overnight with the following antibodies: anti-LC3B (CST no. 2775, 1 : 1000 dilution) or anti-Beclin1 (CST no. 3495, 1 : 1000 dilution). Then, the blots were incubated for 1 h with specific secondary antibodies (1 : 10000, goat anti-rabbit and goat anti-mouse, Molecular Probes, Life Technologies). β-Actin (Sigma-Aldrich) was used as loading control. Immunoreactive bands were detected by chemiluminescence with a Bio-Rad ChemiDoc XRS+ camera. Relative densities were quantified using the Image Lab 4.0 software (Bio-Rad).

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

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