Ab32060
Manufactured by Abcam
Sourced in United Kingdom
Ab32060 is a recombinant anti-BACE1 antibody produced in HEK293 cells. It is designed for use in Western blotting, immunohistochemistry, and ELISA applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab59348, by Abcam (2 mentions)
Ab236874, by Abcam (1 mentions)
Ab76424, by Abcam (1 mentions)
Ab1801, by Abcam (1 mentions)
Image lab software v 4, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse igg secondary antibody, by Bio-Rad (1 mentions)
Ecl detection kit, by Bio-Rad (1 mentions)
Pre stained sds page gels, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Goat anti rabbit igg, by Bio-Rad (1 mentions)
Ab179513, by Abcam (1 mentions)
Ab202554, by Abcam (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Ab32370, by Abcam (1 mentions)
P1379, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Horseradish peroxidase, by GE Healthcare (1 mentions)
Ab109520, by Abcam (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
6 protocols using ab32060
1
Immunohistochemical Analysis of Apoptosis and Proliferation Markers
2
Western Blot Analysis of Apoptotic Markers
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Hela cells were treated with the two compounds PS 1 and PS 2. After treatment, the cells were harvested and lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0). The total protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, USA). The samples were separated electrophoretically using prestained SDS PAGE gels (Bio-Rad Laboratories) and transferred to a polyvinylidene fluoride (PVDF) membranes. The membranes were kept at 4°C overnight in 3% BCA (Sigma-Aldrich) in PBS/Tween-20 and then incubated with the primary antibodies at one of the following: β-actin (ab 1801, Abcam, UK), Bid (ab 32060, Abcam), Bcl-2 (ab 59348, Abcam), caspase-8 (sc-81661, Santa Cruz Biotechnology, USA). After washing with PBS/Tween-20, the membranes were incubated with goat anti-rabbit IgG or with goat anti-mouse IgG HRP conjugated secondary antibodies (Bio-Rad Laboratories). Immunodetection was performed with an enhanced chemiluminescence (ECL) detection kit (Bio-Rad Laboratories). The protein brands were analyzed using ChemiDoc system equipped with Image Lab software v. 4.1 (Bio-Rad Laboratories).
3
Immunoblotting of Apoptosis Regulators
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Briefly, cells (0.4 × 106) were homogenized in ice-cold lysis buffer. After centrifugation at 5,000 g for 30 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 100 µg were used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4561033EDU, Bio-Rad, USA). After blotting, membranes were blocked in 5% skim milk (70,166, Sigma-Aldrich) in tris-buffered saline containing 0.1% Tween-20 (P1379, Sigma-Aldrich). Membranes were incubated with Bcl-2 (1:1,000; ab59348, RRID: AB_289591, Abcam, UK), Bcl-xL (1:1,000; ab32370, RRID: AB_2847960, Abcam, UK), cytochrome c oxidase subunit IV (COX IV) (1:1,000; ab202554, RRID: AB_2847960, Abcam, UK), Bid (1:1,000; ab32060, RRID: AB_365478, Abcam, UK), cytochrome C (1:1,000; #11,940, RRID: AB_2637071, Cell signaling technology, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000; ab8245, RRID: AB_1267174, Abcam, UK) or beta-tubulin (1:1,000; ab179513, RRID: AB_1566837, Abcam, UK), and subsequently with secondary goat anti-rabbit horseradish peroxidase-conjugated antibody. Reaction products were visualized using an enhanced chemiluminescence detection kit (32106, Thermo Scientific, USA) and quantified by densitometry.
4
Western Blot Analysis of Apoptosis Regulators
5
Mapping Poly(A) Sites in BID Gene
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We randomly selected 4 paraffin-embedded tissue sections for immunohistochemical analysis of BID expression in ESCC. The sections were incubated with primary antibody against BID (1:100, Abcam Cat# ab32060, RRID: AB_725689) at 4 C overnight and then detected with the ABC Kit (Pierce).
3 0 -Rapid-amplification of cDNA ends
We used 3 0 -Rapid-amplification of cDNA ends (3 0 -RACE) to determine the poly(A) sites (PAS) of BID with SMARTer RACE 5 0 /3 0 kit (TaKaRa). The sequences of gene-specific primers used for 3 0 -RACE are listed in Supplementary Table S2. The 3 0 -RACE products were then cloned and Sanger sequenced to accurately identify the BID PASs.
3 0 -Rapid-amplification of cDNA ends
We used 3 0 -Rapid-amplification of cDNA ends (3 0 -RACE) to determine the poly(A) sites (PAS) of BID with SMARTer RACE 5 0 /3 0 kit (TaKaRa). The sequences of gene-specific primers used for 3 0 -RACE are listed in Supplementary Table S2. The 3 0 -RACE products were then cloned and Sanger sequenced to accurately identify the BID PASs.
6
Mitochondrial Protein Isolation and Analysis
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A Mitochondria/Cytosol Isolation Kit (BioVision, Inc., Milpitas, CA, USA) was used to separate cytoplasmic and mitochondrial proteins according to the manufacturer's instructions. According to our previous method [14] , the obtained proteins were quantified by the BCA (Thermo Fisher Scientific, Waltham, MA, USA) method. After denaturation, equal amounts of proteins (500 ng/µL, 10 µL) from different samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore). The blots were probed with the following antibodies: BID (ab32060; ABCAm, Cambridge, UK), truncated BID (tBID) (ab108293; ABCAm); cytochrome C (11940; Cell Signaling Technology, Danvers, MA, USA); cleaved caspase-3 (9654; Cell Signaling Technology); cleaved caspase-9 (7237; Cell Signaling Technology); and GAPDH (5174; Cell Signaling Technology). After washing, the membranes were incubated with a secondary antibody. Chemiluminescence (4200; Tanon Science & Technology, Shanghai, China) was used to detect the signals, and ImageJ 1.49v software (National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis of the signals.
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