Antibodies used were mouse anti-USP15 (H00009958-M01, Abnova), anti-TOP2A (sc-165986, Santa Cruz), anti-cyclinB1 (05-373, Millipore), anti-β-actin (ab6276, Abcam), anti-α-tubulin (DM1A, Sigma) and anti-CENPA (ab13939, Abcam); rabbit anti-actin (A2066, Sigma), anti-pericentrin (ab4448, Abcam). Polyclonal affinity-purified sheep anti-GFP was generated in house (IAP).
Ab13939
Manufactured by Abcam
Sourced in United Kingdom
Ab13939 is a laboratory reagent. It is a recombinant protein that can be used in various experimental applications. The core function of this product is to serve as a tool for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
A2066, by Merck Group (2 mentions)
Ab4448, by Abcam (2 mentions)
Ab6276, by Abcam (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Ab15093, by Abcam (2 mentions)
Sc 365189, by Santa Cruz Biotechnology (1 mentions)
P0448, by Agilent Technologies (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Ab4457, by Abcam (1 mentions)
Ab192237, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ab290, by Abcam (1 mentions)
P0449, by Agilent Technologies (1 mentions)
Cytospin, by Hanil (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
21 protocols using ab13939
1
Antibodies for Cell Signaling Analysis
2
Antibody Characterization for IF and IB
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The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).
3
Mitotic HeLa Cell Imaging Protocol
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HeLa cells were treated with 100 ng/ml of nocodazole for 4 h and floating mitotic cells were collected by gentle shake-off. The cells were incubated in KCl (75 mM) buffer for 10 min at room temperature and centrifuged at 200×g for 5 min using Cytospin (Hanil Science, Korea). The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 for 10 min. The fixed cells were blocked with 3% bovine serum albumin for 1 h at room temperature. The antibodies were used; mouse anti-RSF1 (Upstate, 1:100, #05-727), human anti-ACA (Immunovision, Inc, 1:2,000), rabbit anti-H2A K118ac (PTM biology, 1:100, PTM-173), rabbit anti-H2A pT120 (Active motif, 1:500, #39391), mouse anti-CENP-A (abcam, 1:100, ab13939), rabbit anti-POGZ (abcam, 1:100, ab171934), rabbit anti-KAT5/Tip60 (abcam, 1:100, ab23886). For image acquisition, Nikon A1R-A1 Confocal Microscope system with 60× 1.4 NA Plan-Apochromat objective (Nikon Instrument Inc.) or LSM710 with 63× 1.4 NA Plan-Apochromat objective (Carl Zeiss) were used and analyzed by the NIS elements C program or the ZEN 2011 program, and Image processing and quantification were carried out with ImageJ.
4
Immunofluorescence and Western Blot Assays
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Anti-α-tubulin antibody (mouse, FITC-DM1A, F2168; Sigma-Aldrich), anti-CENP-A antibody (mouse, ab13939; Abcam), anti-MKLP1 antibody (rabbit, ab204478; Abcam), and Rhodamine-conjugated phalloidin (R415; Invitrogen) were used for immunofluorescence. The appropriate secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Antibodies used for western blots were anti-α-tubulin (mouse, DM1A, T9017; Sigma-Aldrich) and anti-BubR1 (mouse, 612503; BD Biosciences). Anti-CENP-E antibody (HpX) was generated as previously described (Yao et al., 1997 (link)). Anti-PRC1 antibody used in immunofluorescence and western blot was generated as described (Fu et al., 2007 (link)).
Nocodazole (100 ng/ml), monastrol (50 μM), MG132 (10 μM), and reversine (300 nM) were from Sigma. GSK923295 (100 nM) and BI2536 (100 nM) were from Selleckchem. Syntelin (1 μM) was synthesized as described before (Ding et al., 2010 (link)). The protease inhibitor cocktail was from Sigma.
Nocodazole (100 ng/ml), monastrol (50 μM), MG132 (10 μM), and reversine (300 nM) were from Sigma. GSK923295 (100 nM) and BI2536 (100 nM) were from Selleckchem. Syntelin (1 μM) was synthesized as described before (Ding et al., 2010 (link)). The protease inhibitor cocktail was from Sigma.
5
ChIP Assays for Centromeric DNA
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Chromatin immunoprecipitation (ChIP) assays were performed for all the species using the Pierce Agarose ChIP Kit (Thermo Scientific), following the manufacturer’s recommendations. Immunoprecipitation was carried out using 2 µg of mouse anti-human CENP-A monoclonal antibody (ab13939, Abcam) and normal rabbit Immunoglobulin G (IgG) to control nonspecific binding. One-tenth of starting material was reserved as input DNA control. DNA immunoprecipitated (IP) and input samples were analyzed by a PCR amplification with specific primers for the several satDNA sequences (supplementary table S1 , Supplementary Material online). The input/IP ratio was quantified using Image J software.
6
Quantitative Immunoblotting of Cell Cycle Proteins
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Cells were seeded into 6 cm plates (4 × 105 cells/plate) for 48 h and the lysates were subjected to SDS-PAGE electrophoresis, transferred onto a nitrocellulose membrane, and blocked with 5% skim milk at room temperature. The membranes were incubated with primary antibodies overnight, washed with phosphate buffered saline (PBS), and incubated with anti-mouse (Abcam, ab6728) or rabbit IgG-HRP (Abcam, ab6721). The primary antibodies were as follows: E2F1 (Abcam, ab218527), AURKB (Abcam, ab24), DSN1 (Proteintech, 17,742–1-AP), CCNB2 (Abcam, ab185622), CHEK1 (Abcam, ab40866), CENPA (Abcam, ab13939), DP1 (Abcam, ab124678), β-actin (Santa Cruz Biotechnology, sc69879), and GAPDH (Proteintech, 10,494–1-AP).
7
Chromatin Fractionation and Western Blot
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First, 6 ×106 1BR-hTERT cells were plated into
10 cm dishes and grown for 24 h.
DMSO or 10 μM12 was then added for 48 h, and the
cells were harvested, washed in PBS, and pelleted. Cell pellets were
resuspended in low salt lysis buffer (50 mM Tris pH 8.0, 2 mM EDTA,
2 mM EGTA, 150 mM NaCl, 0.2% Triton-X100, 0.3% NP40) and incubated
on ice for 5 min. Cell lysates were centrifuged at 3000 rpm for 4
min at 4 °C and the supernatant removed as the soluble fraction.
The chromatin pellet was washed three times in ice-cold PBS before
incubation with Benzonase (Sigma) in nuclease buffer (50 mM Tris pH
8.0, 20 mM NaCl, 2 mM MgCl2) for 30 min on ice. An equal
volume of twice the high salt/Triton buffer (1.86 M NaCl, 0.4% Triton-X100)
was added before sonication in a water bath and centrifuged at 14000
rpm for 5 min at 4 °C. The supernatant was retained as the chromatin
fraction. Then 50 μg of protein from the soluble or chromatin
fractions were run on polyacrylamide gels using SDS-PAGE before Western
blotting using antibodies against BAF180 (Bethyl, A301-591A), α-tubulin
(Abcam, ab7291), and CENPA (Abcam, ab13939).
10 cm dishes and grown for 24 h.
DMSO or 10 μM
cells were harvested, washed in PBS, and pelleted. Cell pellets were
resuspended in low salt lysis buffer (50 mM Tris pH 8.0, 2 mM EDTA,
2 mM EGTA, 150 mM NaCl, 0.2% Triton-X100, 0.3% NP40) and incubated
on ice for 5 min. Cell lysates were centrifuged at 3000 rpm for 4
min at 4 °C and the supernatant removed as the soluble fraction.
The chromatin pellet was washed three times in ice-cold PBS before
incubation with Benzonase (Sigma) in nuclease buffer (50 mM Tris pH
8.0, 20 mM NaCl, 2 mM MgCl2) for 30 min on ice. An equal
volume of twice the high salt/Triton buffer (1.86 M NaCl, 0.4% Triton-X100)
was added before sonication in a water bath and centrifuged at 14000
rpm for 5 min at 4 °C. The supernatant was retained as the chromatin
fraction. Then 50 μg of protein from the soluble or chromatin
fractions were run on polyacrylamide gels using SDS-PAGE before Western
blotting using antibodies against BAF180 (Bethyl, A301-591A), α-tubulin
(Abcam, ab7291), and CENPA (Abcam, ab13939).
8
Western Blot Analysis of CENPA and GAPDH
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The cells were pelleted, dissolved in 4× Laemmli buffer, sonicated for 30 s, and immediately placed in ice. Whole-cell extracts were then heated for an additional 2 min at 95 °C and returned to ice. The samples were subsequently separated on 4–20% SDS-polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene fluoride membranes via wet transfer at 80 V for 90 min. The membranes were then incubated in blocking buffer (PBS, 0.1% Tween, 5% nonfat dry milk) for 1 h at room temperature. Primary antibody incubations were conducted with indicated antibodies in blocking buffer at 4 °C overnight. Secondary antibody incubations were conducted the following day with species-appropriate horseradish peroxidase–conjugated secondary antibodies. The blots were developed using enhanced chemiluminescence substrate according to manufacturer's protocol (Millipore). Antibodies against CENPA (Abcam ab13939) and GAPDH (Abcam ab181602) were used as primary antibodies.
9
Protein Extraction and Fractionation
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Cells were pelleted and dissolved in 2% SDS and heated in a boiling water bath for 15 min. Extracts were supplemented with beta-mercaptoethanol and heated for an additional 2 min at 95 °C. Cytoplasmic and nuclear fractions were separated using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fischer scientific) following the protocol described by the manufacturer. Samples were separated using SDS/PAGE, transferred to PVDF membranes, and immunoblotted using, anti-beta actin antibody (Abcam, ab227387), anti-GAPDH antibody (Abcam, ab 9484), anti-Histone H3 (tri methyl K9) antibody (Abcam, ab8898), and anti-CENPA antibody [3-19] (Abcam, ab13939), and the respective secondary antibodies.
10
Comprehensive Chromatin Characterization
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Aurora B (ab2254, Abcam), CENP-A (ab13939, Abcam), CENP-C (PD030, Medical and Biological Laboratories), H3K9me1 (39681, Active Motif), H3K9me2 (05–1249, Millipore), H3K9me3 (07–523, Upstate), H3K4me2 (07–030, Upstate), H3K27me3 (61017, Active Motif & ab6002, Abcam), HA (ab9111, Abcam), HP1α (MAB3584, Millipore), INCENP (ab36453, Abcam).
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