Application of Radio-Immunoprecipitation assay buffer (Beyotime, Shanghai, China) was to extract total protein from cells or tissues. Separation of the same protein samples was via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then electro-blot was onto Polyvinylidene fluoride membrane (Bio-Rad, Inc., Hercules, CA, USA). Then, after seal of the membrane with 5% skimmed milk, incubation was with Bax (1:1,000, ab32503), Bcl-2 (1:1000, ab32124), hypoxia-inducible factor 3 alpha (HIF3A) (1:1,000, ab10134), caspase-3 (1:1,000, ab32351) (Abcam, Cambridge, UK) and GAPDH (1:1000, 2118, Cell Signaling Technology), and secondary antibody (1:5,000, ab6728, Abcam). The immunoblotting was quantified via ImageLab software (Bio-Rad, Inc., Hercules, CA, USA). The protein concentration was normalized to GAPDH.
Ab10134
Manufactured by Abcam
Sourced in United Kingdom
Ab10134 is a primary antibody used for the detection of a specific target protein in biological samples. It is a high-quality research tool designed for use in a variety of laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab32351, by Abcam (2 mentions)
Ab6728, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab16066, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab5694, by Abcam (1 mentions)
Hrp conjugated goat anti mouse, by Santa Cruz Biotechnology (1 mentions)
Ac 15, by Merck Group (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
4 protocols using ab10134
1
Protein Expression Analysis of Cell Lines
2
Western Blot Analysis of HIF Proteins
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Cells or tissues were treated with RIPA lysis buffer (Beyotime, China) to harvest proteins. The concentration of protein was determined using BCA protein assay kit (Thermo Fisher, Waltham, USA). Proteins were resolved by 10% SDS denatured polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membranes were blocked in 5% non-fat milk and incubated with following primary antibodies: anti-HIF3α (Abcam, Cambridge, MA, USA, ab10134, 1:500), anti-HIF1α (Abcam, ab16066, 1:2000) or anti-GAPDH (Abcam, ab8245, 1:5000) overnight at 4°C. Then, the membranes were washed with TBST for 3 times and incubated with HRP-conjugated secondary antibody. The signals of immunoreactive bands were visualized by the enhanced chemiluminescent (ECL) substrate.
3
Western Blot Analysis of Apoptosis-Related Proteins
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Total proteins were extracted from PDLCs using RIPA buffer (Beyotime, Shanghai, China). Equal protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Bio-Rad, Inc., Hercules, CA, USA). Then, the membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies of Bax (1 : 1,000, ab32503, Abcam, Cambridge, UK), Bcl-2 (1 : 1,000, ab32124, Abcam), HIF3A (1 : 1,000, ab10134, Abcam), caspase-3 (1 : 1,000, ab32351, Abcam), and β-actin (1 : 1,000, ab5694, Abcam). After the membranes were washed with tris-buffered-saline Tween (TBST), a secondary antibody (1 : 5,000, ab6728, Abcam) was added to incubate with the membranes at 37°C for 2 h. The immunoblots were quantified by using ImageLab software (Bio-Rad, Inc., Hercules, CA, USA). The relative protein levels of Bax, Bcl-2, HIF3A, and caspase-3 were normalized by β-actin.
4
Western Blot Analysis of HIF Proteins
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Twenty μg of total protein was subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane followed by blocking of the membrane in 5% non-fat dry milk in PBS-Tween (Phosphate buffered saline, 0.05% (v/v) Tween 20) to prevent non-specific antibody binding. Primary and secondary antibody incubations were carried out in the same buffer using anti-HIF1α (610958, mouse monoclonal, 1:500, BD Biosciences, Breda, The Netherlands), anti-HIF3α (ab10134, rabbit polyclonal, 1:500, Abcam, Cambridge, UK) and anti-β-Actin (AC-15, mouse monoclonal, 1:5000, Sigma-Aldrich, Zwijndrecht, The Netherlands) as a loading control. As secondary antibody HRP-conjugated goat-anti-mouse (1:10000, Santa Cruz Biotechnology, Heidelberg, Germany) or goat-anti-rabbit (1:10000, Jackson Immunoresearch, Suffolk, UK) antibodies were used. Antibody incubations were followed by enhanced chemoluminescence (Supersignal West Pico Chemiluminescent Substrate, Thermo Scientific) and visualized on film (Amersham Hyperfilm ECL, GE Healthcare, Diegem, Belgium).
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