Atg3 Antibody

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	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

Protein of whole-cell lysate (4 μg) was reduced and denaturated by boiling in lithium dodecyl sulfate (LDS) sample buffer containing 50 mM dithiothreitol (DTT) at 95 °C for 5 min. Replicate protein samples were separated on Bolt™ 4–12% gradient Bis-Tris gel and blotted onto nitrocellulose membranes using iBlot® 2 Dry Blotting System. Blots were washed once in 1X TBS for 5 min, blocked in 5% w/v skimmed milk for 1 h at room temperature, and probed with the primary antibodies against BiP, calnexin, Ero1α, PDI, IRE1α, CHOP, ATF6 (1:500), CASP12 (1:2000), BID, LC3, ATG3, ATG7, beclin, and β-actin (1:104) overnight at 4 °C. Alternatively, blots were blocked in 5% BSA and probed with the primary antibodies against phospho-IRE1 (Ser724) and phospho-Bcl-2 (Ser70) overnight at 4 °C. All the primary antibodies used were diluted in 5% BSA in 1X TBS with 0.05% Tween® 20 (TBS–T; pH 8) at 1:1000 ratio, unless otherwise specified. All the primary antibodies used were generated in rabbit, except for the antibodies against CHOP, ATF6 and β-actin which were raised in mice. Following primary antibody incubation, blots were washed three times in 1X TBS–T for 5 min each and incubated with the HRP-linked secondary antibodies against the corresponding species IgG (1:2000) for 1 h at room temperature. Blots were developed using Amersham ECL Prime western blotting detection reagent kit as per manufacturer’s instructions. ChemiDoc™ MP Imaging System with Image Lab™ software (BIO-RAD Laboratories Ltd, Hertfordshire, UK) used to image chemiluminescent bands and perform densitometric analysis. β-Actin protein was served as loading control to which relative peak intensities of the examined markers were normalised. To reprobe, blots were incubated in Restore™ PLUS stripping buffer for 20 min at 37 °C with gentle agitation and subsequently washed three times in TBS for 5 min each. Chemiluminescent detection to ensure the removal of the original signal preceded blot re-incubation with another primary antibody of interest.

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Manufacturer protocol

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