Total protein from whole-cell lysate was immunoprecipitated. The extract was incubated by rotating for 3 h in 4°C with protein A/G beads (sc-2003, Santa Cruz, USA). The incubated beads were discarded to clear the nonspecific protein, and 20 μL of the supernatant was taken as input. Then, ATG7 antibody (1 : 500, 8558, Cell Signaling Technology, USA) was added and incubated with rotation for 12 h at 4°C. The beads were centrifuged at 3,000 rpm for 5 min and washed rotating with RIPA lysis buffer (P0013C, Beyotime Biotechnology, China). DTT (ST041, Beyotime Biotechnology, China) was added into the buffer. The beads were resuspended in SDS–PAGE loading buffer (CW0027, CWBIO, China) and then incubated at 99°C for 10 min. The SDS–PAGE electrophoresis test was performed after protein extraction. The immunoreactive bands were incubated with Ac-FOXO1 antibody overnight at 4°C. Protein bands were visualized via an automatic chemiluminescence imaging analysis system (Tanon-5200, USA).
Atg7 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The ATG7 antibody is a laboratory reagent used in research applications. It is an antibody that specifically binds to the ATG7 protein, which plays a crucial role in the autophagy process. The ATG7 antibody can be used to detect and quantify the expression levels of the ATG7 protein in various experimental models and sample types.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Beyotime (1 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Tiangen Biotech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Imagequant las 4000, by Thermo Fisher Scientific (1 mentions)
Tubulin antibody 11224 1 ap, by Proteintech (1 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
Pierce ecl chemiluminescence reagents, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
2 protocols using atg7 antibody
1
Immunoprecipitation and Western Blot Analysis
2
Western Blot Analysis of Autophagy Proteins
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The following antibodies were sued in this study: ATG7 antibody (#8558; Cell Signaling Technology), LC3 antibody (#3868; Cell Signaling Technology), BECN1 antibody (#3738; Cell Signaling Technology), tubulin antibody (11224-1-AP; Proteintech Group, Chicago, IL, USA); caspase-8 antibody (13423-1-AP; Proteintech Group); antirabbit IgG, horseradish peroxidase (HRP)-linked antibody (#7074; Cell Signaling Technology) and anti-mouse IgG, HRP-linked antibody (#7076; Cell Signaling Technology). Cells were lysed with the radioimmunoprecipitation assay (RIPA) buffer (P0013C; Beyotime Biotechnology), and the total protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (PA115; Tiangen Biotech). In addition, proteins from whole-cell extracts were resolved using denaturing SDS-PAGE and analyzed by Western blotting. Subsequently, equal amounts of protein were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). Protein bands were detected with Pierce ECL chemiluminescence reagents (Thermo Fisher) and exposed on the ImageQuant LAS 4000 (Japan) system. Immunoblots shown in figures were derived from three independent experiments. Furthermore, intensities of protein bands were quantified by densitometry using the ImageJ software (http://imagej. nih.gov/ij/).
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