Western blot (WB) analysis was performed to evaluate ANKHD1 expression in both tissues and cells before and after transfection. Proteins were extracted from cells and the protein concentration was determined using a bicinchoninic acid (BCA) kit (Beyotime). After blocking, membranes (Millipore, Boston, MA,USA) were incubated with anti-ANKHD1 antibody (ab117788, Abcam; dilution 1:2000), anti-AKT antibody(AF1777, Biotime; dilution 1:1000), anti p-AKT antibody(AF1546, Biyotime; dilution 1:1000), anti-BAX antibody(ab32503, Abcam; dilution 1:2000), anti-Bcl-2 antibody(ab182858, Abcam; dilution 1:2000) and then incubated overnight at 4 °C. Subsequently, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (ab205718, Abcam; dilution 1:5000) was added to the membranes and incubated for 1 h before detection. All blots derive from the same experiment and were processed in parallel.
Ab117788
Manufactured by Abcam
Sourced in United States
Ab117788 is a lab equipment product offered by Abcam. It is designed to perform a specific laboratory function, but a detailed and unbiased description cannot be provided while maintaining conciseness. Additional information about the core function of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab8580, by Abcam (2 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab205718, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Protein a sepharose beads, by Abcam (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ab187149, by Abcam (1 mentions)
Ab32147, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Minute cytoplasmic nuclear extraction kit, by Invent Biotechnologies (1 mentions)
Ab1791, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
3 protocols using ab117788
1
Western Blot Analysis of ANKHD1 Expression
2
Chromatin Immunoprecipitation (ChIP) Assay
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Whole cell lysate was prepared in IP lysis buffer (Thermo Fisher Scientific) containing 1 × protease/phosphatase inhibitor cocktail (Cell Signaling, Danvers, MA, USA). Total protein (500 μg) was incubated with 1 μg anti-H3K4me3 antibody (ab8580, Abcam, Cambridge, MA, USA) or anti-ANKHD1 antibody (ab117788, Abcam), or rabbit IgG on a rotating shaker at 4 °C for 1 h. Then, protein A Sepharose beads (Abcam) were added to the mixture and shaken at 4 °C overnight. After washing the beads and associated protein complexes three times, they were boiled in 5 × sample loading buffer for 5 min and the supernatant was examined using western blot.
3
Subcellular Fractionation and Western Blot Analysis
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Cytoplasmic and nuclear fractions were prepared from cells using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, USA) according to the manufacturer's instructions.
Whole cell lysates were prepared in RIPA buffer (Thermo Fisher Scientific) containing protease/phosphatase inhibitor cocktail (Cell Signalling). Protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membrane was incubated with the following primary antibodies (all from Abcam): anti-SMYD3 (ab187149, 1:1000), anti-H3K4me3 (ab8580, 1:1000), anti-ANKHD1 (ab117788, 1:2000), anti-CDK2 (ab32147, 1:1000), anti-H3 (ab1791, 1:1000), and anti-GAPDH (ab181602, 1:5000). After washing the membrane with TBST three times, it was incubated with HRP-labelled goat anti-mouse/rabbit IgG (1:5000, Abcam) for 2 h at room temperature. Specific signals were detected using the enhanced chemiluminescence (ECL, Millipore, MA, USA) method and analysed with ImageJ software.
Whole cell lysates were prepared in RIPA buffer (Thermo Fisher Scientific) containing protease/phosphatase inhibitor cocktail (Cell Signalling). Protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membrane was incubated with the following primary antibodies (all from Abcam): anti-SMYD3 (ab187149, 1:1000), anti-H3K4me3 (ab8580, 1:1000), anti-ANKHD1 (ab117788, 1:2000), anti-CDK2 (ab32147, 1:1000), anti-H3 (ab1791, 1:1000), and anti-GAPDH (ab181602, 1:5000). After washing the membrane with TBST three times, it was incubated with HRP-labelled goat anti-mouse/rabbit IgG (1:5000, Abcam) for 2 h at room temperature. Specific signals were detected using the enhanced chemiluminescence (ECL, Millipore, MA, USA) method and analysed with ImageJ software.
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