Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)
Key features and details
- Mouse monoclonal [13D6] to Dicer - ChIP Grade
- Suitable for: WB, ChIP, Flow Cyt
- Knockout validated
- Reacts with: Mouse, Human
- Isotype: IgG2a
Overview
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Product name
Anti-Dicer antibody [13D6] - ChIP Grade
See all Dicer primary antibodies -
Description
Mouse monoclonal [13D6] to Dicer - ChIP Grade -
Host species
Mouse -
Specificity
We do not guarantee its use in WB with mouse lysates. -
Tested Applications & Species
See all applications and species dataApplication Species ChIP HumanFlow Cyt HumanWB MouseHuman -
Immunogen
Synthetic peptide corresponding to Human Dicer.
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Positive control
- WB: 10ng of recombinant dicer (using ECL or ECL Plus) in under one minute of exposure to film. ICC: Human Fibroblasts
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General notes
To our knowledge the localization of Dicer remains to be fully determined and it appears that its localization can be nuclear and/or cytoplasmic. Please see reference Meltzer, 2005 for a reference to a cytoplasmic localisation (figure 1 of paper), as supported with the staining seen in the image on this datasheet.
Please note this antibody gave negative results in WB with mouse fibroblast cell line lysates. We do not guarantee its use in WB with mouse lysates.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
Preservative: 0.02% Sodium azide
Constituent: HEPES buffered saline -
Concentration information loading...
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Purity
Ammonium Sulphate Precipitation -
Clonality
Monoclonal -
Clone number
13D6 -
Isotype
IgG2a -
Research areas
Images
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Dicer knockout HAP1 cell lysate (20 µg)Lanes 1 and 2: Merged signal (red and green). Green - ab14601 observed at 240 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab14601 was shown to specifically react with Dicer in wild-type HAP1 cells. No band was observed when Dicer knockout samples were used. Wild-type and Dicer knockout samples were subjected to SDS-PAGE. ab14601 and ab181602 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging. -
All lanes : Anti-Dicer antibody [13D6] - ChIP Grade (ab14601) at 1/2000 dilution (incubated overnight at 4°C.)
Lane 1 : NIH 3T3
Lane 2 : A549
Lane 3 : HepG2
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Mouse Green at 1/10000 dilutionThis blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab14601 overnight at 4°C. Antibody binding was detected using IR-labelled goat anti-mouse Ab at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
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Overlay histogram showing HEK293 cells stained with ab14601 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab14601, 0.1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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HepG2 cell lysate at 20 µg
Developed using the ECL technique.
Performed under reducing conditions.
Additional bands at: 100 kDa (possible non-specific binding), 60 kDa (possible non-specific binding)
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ChIP analysis of Dicer protein binding at chicken ß-globin regulatory elements. Chromatin from human erythroleukemia (K562) cells containing chicken chromosomes with normal (FRK2) or mutant (HS1-K) ß-globin loci and chicken DT40 cells was immunprecipitated with antibodies to Dicer (ab14601). PCR analysis of immunoprecipitated chromatin was carried out using primers to the chicken ß-globin regulatory elements. Mouse IgG served as control. 10% of input DNA was utilized.
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All lanes : Anti-Dicer antibody [13D6] - ChIP Grade (ab14601) at 2.5 µg/ml
Lane 1 : human B cell lymphoma lysate
Lane 2 : human B cell lymphoma lysate infected with control shRNA retrovirus
Lane 3 : human B cell lymphoma lysate infected with Dicer knockdown shRNA virus
Lysates/proteins at 2.5 µg/ml per lane.
Observed band size: 225 kDa why is the actual band size different from the predicted?