Atg5 (D5F5U) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100. Incubate for 16 h at 4 °C

Protocol tips
For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100. Incubate for 16 h at 4 °C

Publication protocol

At the end of treatment, plates were placed on ice and the cell monolayers rinsed with PBS, lysed and processed as described previously [23]. Briefly, cells were collected by gentle centrifugation, the cell pellet rinsed in PBS and then re-suspended in lysis buffer. Cell lysates were cleared by centrifugation at 16,000× g for 20 min at 4 °C and the protein concentration determined using the bicinchoninic acid (BCA) (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell lysates were adjusted for protein content and equal amounts of total protein were separated by SDS-PAGE and immobilized onto PVDF membranes. Membranes were then blocked in 5% (w/v) non-fat dry milk in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl (TBS) plus 0.1% Tween-20 (TBS-Tween), and subsequently incubated for 16 h at 4 °C with the appropriate primary antibody in 5% (w/v) bovine serum albumin (BSA). The following day, membranes were washed in TBS-Tween (3 × 10 min) and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Finally, membranes were washed in TBS-Tween (4 × 5 min), and proteins revealed using an enhanced chemiluminescence (ECL) reagent

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Atg5 (D5F5U) Rabbit mAb below.

Download manufacturer protocol

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