Overview

  • Product name
  • Description
    Rabbit polyclonal to HDAC2
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, IHC-FrFl, ICC/IF, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, African green monkey
    Does not react with: Chicken
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 450 to the C-terminus of Human HDAC2.

    (Peptide available as ab16200.)

  • Positive control
    • WB: HAP1, HeLa, A431, Jurkat and HEK293 whole cell lysates and HeLa nuclear lysate. ICC/IF: HDAC2 wildtype cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab16032 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr 1/500.
IHC-P 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-FrFl Use at an assay dependent concentration. PubMed: 23469282
ICC/IF Use a concentration of 0.5 µg/ml.
WB Use a concentration of 0.5 µg/ml. Detects a band of approximately 60 kDa (predicted molecular weight: 55.3 kDa).
IP Use at an assay dependent concentration.

Target

  • Function
    Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes.
    Forms transcriptional repressor complexes by associating with MAD, SIN3, YY1 and N-COR. Interacts in the late S-phase of DNA-replication with DNMT1 in the other transcriptional repressor complex composed of DNMT1, DMAP1, PCNA, CAF1. Deacetylates TSHZ3 and regulates its transcriptional repressor activity.
  • Tissue specificity
    Widely expressed; lower levels in brain and lung.
  • Sequence similarities
    Belongs to the histone deacetylase family. HD type 1 subfamily.
  • Post-translational
    modifications
    S-nitrosylated by GAPDH. In neurons, S-Nitrosylation at Cys-262 and Cys-274 does not affect the enzyme activity but abolishes chromatin-binding, leading to increases acetylation of histones and activate genes that are associated with neuronal development. In embryonic cortical neurons, S-Nitrosylation regulates dendritic growth and branching.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • D10Wsu179e antibody
    • HD 2 antibody
    • HD2 antibody
    • HDAC 2 antibody
    • Hdac2 antibody
    • HDAC2_HUMAN antibody
    • Histone deacetylase 2 (HD2) antibody
    • Histone deacetylase 2 antibody
    • OTTHUMP00000017046 antibody
    • OTTHUMP00000227077 antibody
    • OTTHUMP00000227078 antibody
    • RPD3 antibody
    • transcriptional regulator homolog RPD3 antibody
    • YAF1 antibody
    • YY1 associated factor 1 antibody
    • YY1 transcription factor binding protein antibody
    • Yy1bp antibody
    see all

Images



  • Predicted band size : 55.3 kDa

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: HDAC2 knockout HAP1 whole cell lysate (20 µg)

    Lanes 1 - 2: Merged signal (red and green). Green - ab16032 observed at 60 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab16032 detected the expected band for HDAC2 in wild-type HAP1 cells and the band was not seen in HDAC2 knockout HAP1 cells. Additional cross-reactive bands were detected. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  Ab16032 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • ab16032 staining HDAC2 in wild-type HAP1 cells (top panel) and HDAC2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16032 at 0.5μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Immunohistochemistry - Free Floating analysis of mouse brain labelling HDAC2 with ab16032 at 1/250 dilution. HDAC2 was detected in the nuclei (arrows) of dopamine neurons, neuronal marker (magenta) and Hoechst stain (blue).

    Brains were post-fixed overnight in phosphate-buffered 4% PFA, and equilibrated in 30% sucrose for 2 days. Brains were sectioned on a cryostat at 30 µm. Sections were stored in a cryoprotective tissue collection solution (25% glycerol, 30% ethylene glycol, 0.05 M phosphate buffer (PB)) at −20°C until use. Immunofluorescence was performed using a free-floating method.

  • Immunohistochemistry - Free Floating analysis of mouse brain labelling HDAC2 with ab16032 at 1/250 dilution. HDAC2 was detected in the nuclei (arrows) of histamine neurons, neuronal marker (magenta) and Hoechst stain (blue).

    Brains were post-fixed overnight in phosphate-buffered 4% PFA, and equilibrated in 30% sucrose for 2 days. Brains were sectioned on a cryostat at 30 µm. Sections were stored in a cryoprotective tissue collection solution (25% glycerol, 30% ethylene glycol, 0.05 M phosphate buffer (PB)) at −20°C until use. Immunofluorescence was performed using a free-floating method.

  • ICC/IF image of ab16032 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab16032 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • All lanes : Anti-HDAC2 antibody (ab16032) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 55.3 kDa
    Observed band size : 60 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 50 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 minute
  • HDAC2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5ug of Rabbit polyclonal to HDAC2 and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Hela whole cell extractdiluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16032. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 60ka: HDAC2.
  • ab16032 (1/250) staining HDAC2 in paraffin-embedded Human tonsil tissue.  Tissue underwent fixation in formaldehyde, peroxidase blocking, protein blocking and heat mediated antigen retrieval.  The secondary antibody was goat anti rabbit/mouse conjugated to HRP.  For further experimental details please refer to abreview.

    See Abreview

  • IHC-Fr image of HDAC2 (ab16032) staining on Rat spinal cord. The sections required antigen retrieval with sodium citrate buffer was necessary to obtain full signal strength (sodium citrate 10mM, pH6)

    See Abreview

  • All lanes : Anti-HDAC2 antibody (ab16032) at 0.4 µg/ml

    Lane 1 : Mouse 3T3 lysate
    Lane 2 : Rat liver lysate
    Lane 3 : Chicken liver lysate

    Lysates/proteins at 20 µg/ml per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 55.3 kDa
    Observed band size : 60 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 50 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 30 secondsab16032 cross-reacts with Mouse 3T3 cells but shows no cross-reactivity against either Rat liver or Chicken cell lysates.

References

This product has been referenced in:
  • Pardo M  et al. Intranasal siRNA administration reveals IGF2 deficiency contributes to impaired cognition in Fragile X syndrome mice. JCI Insight 2:e91782 (2017). Read more (PubMed: 28352664) »
  • Bednarczyk J  et al. MBD3 expression and DNA binding patterns are altered in a rat model of temporal lobe epilepsy. Sci Rep 6:33736 (2016). WB ; Rat . Read more (PubMed: 27650712) »

See all 11 Publications for this product

Customer reviews and Q&As

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (human breast cancer tissue sections)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 6.0 citric acid
Permeabilization
Yes - tween-20
Specification
human breast cancer tissue sections
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Nov 17 2015

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (primary culture cell of human breast carcinoma tis)
Permeabilization
Yes - tween-20
Specification
primary culture cell of human breast carcinoma tis
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Nov 17 2015

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (hippocampus)
Specification
hippocampus
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Miss. Sung-Eun WANG

Verified customer

Submitted Apr 17 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
22 µg
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Sample
Mouse Cell lysate - whole cell (293T, MEL cell)
Specification
293T, MEL cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 03 2013

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Sample
African Green Monkey Cell (Cos7 Monkey Kidney Fibroblast)
Specification
Cos7 Monkey Kidney Fibroblast
Permeabilization
Yes - .3% Triton X100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 13 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (spinal cord)
Specification
spinal cord
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Sep 06 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tonsil)
Specification
Tonsil
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Dako Target Retrieval Solution
Permeabilization
No
Blocking step
5 minutes of peroxidase block then 10 minutes of protein block. These are ready-to-use reagents purchased from Dako as blocking agent for 15 minute(s) · Concentration: 100% · Temperature: 20°C
Username

Mr. Antibody Solutions

Verified customer

Submitted Mar 25 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Human colon adenocarcinoma cells)
Loading amount
200000 cells
Specification
Human colon adenocarcinoma cells
Treatment
transfected with plasmids (irrelevant to HDAC2)
Gel Running Conditions
Reduced Denaturing (12% gel)
Blocking step
Milk as blocking agent for 40 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Ms. Johanna Smith

Verified customer

Submitted Jul 11 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (various cell types - cell lines and human primary)
Loading amount
15 µg
Specification
various cell types - cell lines and human primary
Gel Running Conditions
Reduced Denaturing (10% gel)
Blocking step
Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Prof. Paul Townsend

Verified customer

Submitted May 01 2008

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