Recombinant
RabMAb

Recombinant Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade (ab51997)

Overview

  • Product name

    Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade
    See all Histone H4 primary antibodies
  • Description

    Rabbit monoclonal [EP1000Y] to Histone H4 (acetyl K5) - ChIP Grade
  • Host species

    Rabbit
  • Specificity

    In addition to H4K5Ac, this antibody also detects H4K8Ac (Histone H4 acetylated on Lysine 8) at high antigen coating concentration.
  • Tested applications

    Suitable for: ChIP, ELISA, WB, IHC-P, ICC/IF, IPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human, Xenopus laevis, Rice
  • Immunogen

    Synthetic peptide within Human Histone H4 aa 1-100 (N terminal) (acetyl K5). The exact sequence is proprietary.
    Database link: P62805

  • Positive control

    • HeLa, NIH/3T3, C6 cells or human brain glioma, human cervical carcinoma, human normal colon FFPE, mouse liver and rat cerebral cortex tissue. ChIP: Chromatin was prepared from MEF cells.
  • General notes

    A trial size is available to purchase for this antibody.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab51997 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
ChIP Use 5 µg for 25 µg of chromatin.
ELISA Use at an assay dependent concentration.
WB 1/500000. Detects a band of approximately 11 kDa (predicted molecular weight: 11 kDa).

For unpurified use at 1/10000- 1/50000.

IHC-P 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF 1/5000.

For unpurified use at 1/250- 1/500.

IP 1/30.

Target

Images

  • Chromatin was prepared from MEF (Mouse embryonic fibroblast cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab51997 (red), and 20 μl protein A/G sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

  • All lanes : Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade (ab51997) at 1/500000 dilution (purified)

    Lane 1 : Nuclear extract of HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 7mM Sodium Butyrate for 24 hours
    Lane 2 : Untreated nuclear extract of HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysates
    Lane 4 : Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysates
    Lane 5 : C6 (Rat glial tumor cell line) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysates
    Lane 6 : Untreated C6 (Rat glial tumor cell line) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 11 kDa
    Observed band size: 11 kDa



    Blocking and diluting buffer: 5% NFDM/TBST.

  • IHC image of unpurified ab51997 staining Histone H4 (acetyl K5) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51997, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma)treated with 500ng/m Trichostatin A for 4 hours labeling Histone H4 (acetyl K5) with purified ab51997 at 1/5000 dilution (0.1μg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab195889, an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain at 1/200 (2.5 μg/ml). ab150077, a Goat anti rabbit IgG(Alexa Fluor® 488) secondary antibody was used at 1/1000 dilution. PBS instead of the primary antibody was used as a control. DAPI nuclear staining.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

  • Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 10ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

  • Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 100ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

  • Direct ELISA with Histone H4 K5ac peptide, Histone H4 K8ac peptide, Histone H4 K16me1 peptide, Histone H4 K16me3 peptide, and Histone H4 unmodified peptide, all at 1000ng/ml. ab51997 used as the primary antibody at a range of 0~1000ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) used as the secondary antibody at 1:2500 dilution.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue sections labeling Histone H4 (acetyl K5) with purified ab51997 at 1/500 dilution (1 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Hematoxylin was used to counter stain. ab97051, a Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1/500 dilution. PBS instead of the primary antibody was used as the negative control.

  • All lanes : Anti-Histone H4 (acetyl K5) antibody [EP1000Y] - ChIP Grade (ab51997) at 1/1000000 dilution (unpurified)

    Lane 1 : Untreated HeLa cells
    Lane 2 : TSA treated HeLa calls

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit HRP labelled (1:2000)

    Predicted band size: 11 kDa
    Observed band size: 11 kDa

  • ab51997 (purified) at 1/30 dilution (2µg) immunoprecipitating Histone H4 (acetyl K5) in HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate.

    Lane 1 (input): HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate 10ug
    Lane 2 (+): ab51997+ HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51997 in HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with TSA whole cell lysate

    For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/10000).

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  • ICC/IF image of unpurtified ab51997 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51997, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:

See all 53 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
ChIP
Sample
Human Cell lysate - nuclear (MCF7 breast cancer cell line)
Specification
MCF7 breast cancer cell line
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde

Dr. Sankari Nagarajan

Verified customer

Submitted Apr 12 2019

Application
ChIP
Sample
Arabidopsis thaliana Tissue lysate - nuclear (complete seedlings (10 days old))
Negative control
IgG (included in the Abcam kit)
Specification
complete seedlings (10 days old)
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Positive control
actin

Abcam user community

Verified customer

Submitted Dec 05 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (THP-1)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
THP-1
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Aliaksandra Radzisheuskaya

Verified customer

Submitted Oct 17 2018

Application
ChIP
Sample
Human Cell lysate - whole cell (THP-1 cells)
Negative control
no antibody control, gene desert region
Specification
THP-1 cells
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Positive control
active gene promoter

Aliaksandra Radzisheuskaya

Verified customer

Submitted Oct 17 2018

Application
Flow Cytometry
Sample
Mouse Cell (Thymus)
Permeabilization
Yes - FIX & PERM® Cell Fixation & Cell Permeabilization Kit
Gating Strategy
Thymocytes (CD45+ Epcam- cells)
Specification
Thymus
Fixation
FIX & PERM® Cell Fixation & Cell Permeabilization Kit

Abcam user community

Verified customer

Submitted May 20 2015

Answer

We listed ChIP on the datasheet based on a reference that used the antibody for this application.

Knutson SK et al. Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks. EMBO J 27:1017-28 (2008).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323257/

The authors do not give much detail of their procedure except this:

The ChIP assay was performed following the standard protocol of the Chromatin Immunoprecipitation Assay Kit from Upstate Cell Signaling, with slight modifications using minced tissue for formaldehyde crosslinking. DNA was purified using the Qiagen PCR Purification Kit. Primers designed to the designated promoter sequences were used for quantification of histone acetylation using Q-PCR, with histone H3 and input sonicated DNA as reference controls.

I suggest testing a range of amounts. Our general recommendation is 1-10ug of antibody per 25ug of DNA. Here is a link to our general cross-linked ChIP protocol:

https://www.abcam.com/index.html?pageconfig=resource&rid=11698.

We also have a ChIP kit of our own, ab500.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Xenopus laevis Cell (Fibroblast)
Specification
Fibroblast
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Apr 05 2013

Application
Western blot
Sample
Schizosaccharomyces pombe Purified protein (Purified modified histone peptides)
Loading amount
0.1 µg
Specification
Purified modified histone peptides
Gel Running Conditions
Non-reduced Non-Denaturing (Native)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Feb 03 2010

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