The protein extraction from brain tissues or bEnd.3 cells was quantified by BCA protein assay kit (Beyotime Biotechnology, Shanghai, China), denatured at 98 °C for 10 min, and then, separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against B-cell lymphoma-2 (Bcl-2) (ab194583, 1 : 1000 dilution, Abcam, Cambridge, UK), Bcl-2-associated x protein (Bax) (ab182733, 1 : 2000 dilution, Abcam), FOXO3 (ab70315, 1 : 5000 dilution, Abcam) and β-actin (ab8227, 1 : 5000 dilution, Abcam). Subsequently, the membranes were incubated with secondary antibody (ab6721, 1 : 10 000 dilution, Abcam), conjugated by horseradish peroxidase for 2 h at room temperature and interacted with enhanced chemiluminescence chromogenic substrate (Beyotime Biotechnology). The protein signals were analyzed with Image Lab software (Bio-Rad) with β-actin as the loading control.
Ab70315
Manufactured by Abcam
Sourced in United Kingdom
Ab70315 is a lab equipment product. It has a core function of [insert concise, factual, and unbiased description of the product's core function, without interpretation or extrapolation].
Automatically generated - may contain errors
Lab products found in correlation
Ab182733, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence chromogenic substrate, by Beyotime (1 mentions)
Ab194583, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab8227, by Abcam (1 mentions)
Glycine solution, by Cell Signaling Technology (1 mentions)
Pcr clean up kit, by Beyotime (1 mentions)
Ab172730, by Abcam (1 mentions)
Te buffer, by Beyotime (1 mentions)
Protein a g beads, by Beyotime (1 mentions)
2 protocols using ab70315
1
Western Blot Analysis of Apoptosis Regulators
2
ChIP-Seq for FOXO3 Binding
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The 4 × 106 cells were incubated with 1% fresh formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature and with 2 mL glycine solution (Cell Signaling Technology) for 5 min at room temperature. After the centrifugation at 2000× g for 5 min at 4 °C, cell pellets were incubated with micrococcal nuclease for 20 min at 37 °C to obtain DNA. After the purification procedure, DNA was incubated with protein A/G beads (Beyotime) binding to anti-FOXO3 (ab70315, 2.5 μg/mg, Abcam) or anti-IgG (ab172730, 0.5 μg, Abcam) overnight at 4 °C. Beads were collected to be washed by low salt immune complex wash buffer (Beyotime), high salt immune complex wash buffer (Beyotime), LiCl immune complex wash buffer (Beyotime) and TE buffer (Beyotime), successively. The enrichment and purification of DNA was performed by a PCR clean up kit (Beyotime). DNA expression was quantified by qPCR.
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