ATG5 Antibody (N-term)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Homoginize samples in hypotonic buffer.	- Dilute Primary Ab at -1:1000.

Upstream tips
- Homoginize samples in hypotonic buffer.
Protocol tips
- Dilute Primary Ab at -1:1000.

Publication protocol

Retina and RPE/choroid samples were dissected from the experimental groups. Samples were homogenized in hypotonic buffer, and the proteins separated by SDS-PAGE (4%–15% TrisHCl Ready Gels; Bio-Rad Laboratories, Hercules, CA, USA) and blotted on polyvinylidene fluoride membranes (Immobilon-P; Millipore, Billerica, MA, USA). The membranes were incubated overnight with primary antibodies according to the manufacturer’s instructions. The following antibodies were used: LC3 and Atg12 (1:1000; Cell Signaling), Atg5 (1:1000; Abgent, San Diego, CA, USA), and GAPDH (1:30,000; Invitrogen). Secondary antibodies were from Dako (Dako, Glostrup, Denmark). Detection was by SuperSignal West Dura Substrate (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ protocols. Quantitative densitometry of the immunoblots was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and expressed as the mean density (6SD) from replicate animal groups. All experiments were performed a minimum of three times. Consisten outcomes were obtained and representative experiments are shown.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Abgent for ATG5 Antibody (N-term) below.

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