ATG5 antibody [EPR1755(2)]

Protocol tips

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	Dilute primary antibody at 1:1,000 dilution and incubate overnight at 4°C

Protocol tips
Dilute primary antibody at 1:1,000 dilution and incubate overnight at 4°C

Publication protocol

Cell lysis and western blot analysis
Following SJKJT treatment, the HepG2 cells were washed with ice-cold PBS. The cells were lysed in 1X RIPA lysis buffer, containing protease inhibitors. The cells were then removed and collected into eppendorf tubes (Quality Scientific Plastics, Inc., San Diego, CA, USA), which were agitated for 30 min at 4°C, followed by centrifugation at 13,000 × g for 10 min at 4°C (5415D; Eppendorf, Hamburg, Germany). The protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal quantities of sample (10 µg/lane) were loaded into wells containing 6–10% SDS-polyacrylamide gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and were separated by SDS-PAGE. The separated proteins were then electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore) at 400 mA for 2 h. The membranes were then incubated in blocking buffer (PBS with 0.05% Tween 20 and 5% non fat dry-milk) for 1 h at room temperature, followed by incubation with the following primary antibodies overnight at 4°C: Rabbit polyclonal Beclin-1 (cat. no. 3738); rabbit polyclonal LC3B (cat. no. 2775); rabbit monoclonal p62 (cat. no. 8025); rabbit polyclonal phosphorylated (p)-PI3K (cat. no. 4228); rabbit polyclonal PI3K (cat. no. 4292); rabbit polyclonal p-mTOR (cat. no. 2971); rabbit monoclonal mTOR (cat. no. 2983); rabbit polyclonal p-Akt (cat. no. 9275); rabbit polyclonal Akt (cay. no. 9272); rabbit monoclonal p-ERK1/2 (cat. no. 4370); rabbit monclonal ERK1/2 (cat. no. 4695); rabbit monoclonal p-SAPK/JNK (cat. no.4668); rabbit polyclonal SAPK/JNK (cat. no. 9252); rabbit monoclonal p-p38 (cat. no. 4511); rabbit polyclonal p38 (cat. no. 9212) (all Cell Signaling Technology, Inc., Danvers, MA, USA); rabbit monoclonal Atg-3 (cat. no. GTX63041); and rabbit monoclonal Atg-5 (cat. no. GTX62601; both GeneTex, Inc., Irvine, CA, USA) and mouse monoclonal β-actin (cat. no. A5441; Sigma-Aldrich. All primary antibodies were used at 1:1,000 dilutions. The membranes were then incubated with HRP-conjugated goat anti-rabbit (1:10,000; cat. no. AP132P) and goat anti-mouse (1:10,000; cat. no. AP124P) secondary antibodies (Merck Millipore) for 1 h at room temperature. The blots were washed three times in 1X PBS-Tween 20 solution and incubated for 1 min with WesternBright Quantum enhanced chemiluminescence reagents. The results were visualized by exposing the blots to Super RX-N film (Fujifilm Corporation, Tokyo, Japan). The protein expression levels were quantified using Image J software (1.42q; National Institutes of Health, Bethesda, MD, USA, 2009).

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