Ab5070
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab5070 is a lab equipment product offered by Abcam. It is designed for general laboratory use without any specific intended application.
Automatically generated - may contain errors
Lab products found in correlation
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Ab817, by Abcam (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Anti mouse igg hrp, by Bio-Rad (1 mentions)
Anti gfp antibody, by Takara Bio (1 mentions)
Anti h3k27ac, by Active Motif (1 mentions)
Anti flag m2 peroxidase, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti h3k18ac, by Active Motif (1 mentions)
Chemi lumi one, by Nacalai Tesque (1 mentions)
Anti h4k12ac, by Active Motif (1 mentions)
Anti psi antibody, by Biomatik (1 mentions)
Anti psi, by Biomatik (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Ab97051, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Ab156870, by Abcam (1 mentions)
Ab108203, by Abcam (1 mentions)
7 protocols using ab5070
1
Immunoprecipitation of RNA Pol II
2
Western Blot Analysis of RNAi Pathway Proteins
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Protein samples were denatured by boiling at 95°C for 3 min in protein-loading buffer [2% (w/v) SDS, 100 mM DTT, 0.05% (v/v) BPB, and 10% (v/v) glycerol], resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 4% (w/v) skim milk (Nacalai) in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody (1:500; guinea pig) (80 (link)), anti-GFP antibody (1:2000; Clontech, rabbit), anti-Ago1 antibody (1:1000; Abcam, Ab5070, rabbit), anti-Ago2 antibody (1:100; guinea pig) (25 (link)), anti-Piwi (1:10; mouse, P4D2), anti-FLAG M2-peroxidase [horseradish peroxidase (HRP)] (1:5000; Sigma-Aldrich, #A8592), anti-H3K18Ac (1:2000; Active Motif, #39756), anti-H3K27Ac (1:2000; Active Motif, #39136), anti-H4K8Ac (1:2000; Active Motif, #61104), anti-H4K12Ac (1:2000; Active Motif, #39928), anti-guinea pig immunoglobulins-HRP (1:1000; Dako), anti-rabbit immunoglobulin G (IgG)–HRP (1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio-Rad). The chemiluminescent signals were obtained by using Chemi-Lumi One (Nacalai) and detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed by using Pixelmator or Fiji. The immunoblot signals were quantified by using Fiji.
3
Fly lysate preparation and miRNA detection
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Fly lysate was prepared from 3-d and 30-d male Flag-HA-Ago2 whole flies (Czech et al. 2008 (link)). Approximately 80 flies were used for each IP (Ago1 or Ago2) followed by Northerns (miR-34-5p, esi-2.1, miR-305-5p, miR-263a-5p, miR-11-3p, and miR-317-3p). Immunoprecipitation was performed as described (Kirino et al. 2011 (link)), except that 800 mM NaCl (final concentration) was used for Ago2-IP. Anti-Ago1 (ab5070, Abcam) and M2 beads (#A2220, Sigma-Aldrich) were used for Ago1 and Ago2-IP, respectively. After extraction of RNA from beads as described in Kirino et al. (2011) (link), the purified RNA was loaded onto 15% TBE-urea gel followed by Northern blots.
4
Co-immunoprecipitation of AGO1 and Psi
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Co-IP was performed using 25 wild-type third instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1× protease inhibitor cocktail). Following homogenisation, protein was collected by centrifugation at 13,000 g for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose beads (GE Healthcare Life Science) for 1 h at 4°C with rotation and the supernatant collected by centrifugation at 13,000 g . Equal amounts of pre-cleared protein lysate were incubated with either anti-AGO1 antibody (Abcam, ab5070, 1:70), anti-Psi antibody (custom generated rabbit polyclonal antibody, Biomatik, 1:100) or without antibody (mock IP control) overnight at 4°C. Beads were washed five times with lysis buffer and the eluent resolved using 10% SDS PAGE/western blot with appropriate primary antibody before detection with Li-Cor Odyssey IR.
5
Co-immunoprecipitation of AGO1 and Psi Proteins
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Co-Immunoprecipitation (Co-IP) was performed using 25 wild type 3rd instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1x Protease inhibitor cocktail). Following homogenization, protein was collected by centrifugation at 12 000 rpm for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose TM beads (GE Healthcare Life Science) for 1 hour at 4°C with rotation and the supernatant collected by centrifugation at 12 000 rpm. Equal amounts of pre-cleared protein lysate were incubated with either anti-AGO1 (Abcam, ab5070) or anti-Psi (custom generated rabbit polyclonal antibody, Biomatik) antibodies overnight at 4°C. Beads were washed with lysis buffer 5 times, and the eluent resolved using 10% SDS PAGE/Western with appropriate primary antibody prior to detection with Li-Cor Odyssey IR detection.
6
miRNA Ribonucleoprotein Immunoprecipitation
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Immunoprecipitation of miRNA ribonucleoprotein (miRNP) with anti-Ago1 (1:3,000; cat. no. ab5070; Abcam, Cambridge, UK) or IgG (1:2,000; cat. no. ab97051; Abcam) was performed as previously described (29) . The RNA that was immunoprecipitated with anti-Ago1 or IgG antibodies was extracted using TRIzol LS (Invitrogen; Thermo Fisher Scientific, Inc.) as described previously (30) .
7
Comparative Protein Expression Analysis
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Protein samples were extracted from cells by using RIPA buffer (Thermo Scienti c) supplemented with the protease inhibitor cocktail (MedChemExpress, NJ, USA). Total protein concentrations were quantitated via the BCA method (Thermo Scienti c), and then equal amounts of proteins samples were subjected to SDS-PAGE, followed by immunoblotting. Primary antibodies anti-ADH1A (ab108203), anti-ADH1B (ab175515), anti-ADH1C (ab168748), anti-ADH4 (ab137077), anti-ADH5 (ab177932), anti-AGO1 (ab5070), anti-AGO2 (ab156870) were purchased from Abcam (Cambridge, MA, USA). Anti-ADH6 was purchased from Santa Cruz (CA, USA), and anti-β-Actin (AC-15, Boster, Wuhan, China) was used as a control antibody. After HRP-conjugated secondary antibody incubation, the target band was detected using the chemiluminescence system (Tanon 5200, Shanghai.China).
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