The obtained cell proteins were resolved by SDS-PAGE. After transferring, the membranes were blocked in 5% defatted milk (Bio-Rad) and then incubated with the primary antibodies: anti-LAMP2 (1:500 dilution, Santa Cruz Biotechnology, United States), Cathepsin D (1:1,000 dilution, Cell Signaling Technology, United States), LC3B (1:1,000 dilution, Cell Signaling Technology, United States), ATG5 (1:1,000 dilution, Proteintech, United States) and β-actin (1:10,000 dilution, Multisciences, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies (Multisciences, China). Immunoblots were visualized by ECL substrate (Biosharp) and Bio-Rad Image Lab software.
Defatted milk
Manufactured by Bio-Rad
Sourced in United States
5% defatted milk is a laboratory reagent composed of defatted milk powder dissolved in water to a concentration of 5%. It is commonly used as a blocking agent in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to reduce non-specific binding of antibodies to the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cathepsin d, by Cell Signaling Technology (1 mentions)
Anti lamp2, by Santa Cruz Biotechnology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by MultiSciences Biotech (1 mentions)
β actin, by MultiSciences Biotech (1 mentions)
Ab200708, by Abcam (1 mentions)
Ab210498, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bcl 2, by Thermo Fisher Scientific (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab56416, by Abcam (1 mentions)
Tfap2c, by Merck Group (1 mentions)
Ma5 11757, by Thermo Fisher Scientific (1 mentions)
Bca protein quantitation kit, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
2 protocols using defatted milk
1
Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of Protein Markers
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We prepared protein lysates using RIPA lysis buffer. Protein concentration was tested with BCA Protein Quantitation Kit (Beyotime, Shanghai, China). Protein aliquots were separated through 10–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA), which were blocked using 5% defatted milk (Bio-Rad, Hercules, CA, USA) under ambient temperature for 1 hour and incubated using primary antibodies overnight under 4°C: TFAP2C (AV38284, 1:1,000; Sigma), DUSP5 (ab200708; 1:1,000; Abcam), p62 (ab56416, 1:1,000; Abcam), LC3 (#4108, 1:1,000; Cell Signaling Technology), Beclin1 (ab210498, 1:1,000; Abcam), Bax (ab182733, 1:2,000; Abcam), Bcl-2 (MA5-11757, 1:1,000; Invitrogen), GAPDH (ab9485, 1:1,000; Abcam), followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (#7074, 1:1,000; Cell Signaling Technology) for 1 hour at room temperature. Signals were visualized using SuperSignal Pico PLUS chemiluminescent substrate (Pierce, Rockford, IL, USA). GAPDH served as a loading control. Data analysis was conducted using ImageJ software (NIH, Bethesda, MD, USA).
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