Recombinant Anti-Histone H3 (phospho S28) antibody [E191] - BSA and Azide free (ab215532)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E191] to Histone H3 (phospho S28) - BSA and Azide free
- Suitable for: ChIP, Flow Cyt (Intra), Dot blot, ICC/IF, IHC-P, WB, IP
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3 (phospho S28) antibody [E191] - BSA and Azide free
See all Histone H3 primary antibodies -
Description
Rabbit monoclonal [E191] to Histone H3 (phospho S28) - BSA and Azide free -
Host species
Rabbit -
Specificity
This antibody detects Histone H3 and Histone H3.3 when phosphorylated on Serine 28. It does not detect H3.3 when phosphorylated on Serine 31.
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Tested applications
Suitable for: ChIP, Flow Cyt (Intra), Dot blot, ICC/IF, IHC-P, WB, IPmore details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat, Guinea pig -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- NIH 3T3 whole cell lysate (ab7179) can be used as a positive control in WB.
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General notes
ab215532 is the carrier-free version of ab32388.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.2
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E191 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Alexa Fluor® 488 Anti-Histone H3 (phospho S28) antibody [E191] (ab237417)
- Alexa Fluor® 647 Anti-Histone H3 (phospho S28) antibody [E191] (ab237418)
- PE Anti-Histone H3 (phospho S28) antibody [E191] (ChIP Grade) (ab303149)
- APC Anti-Histone H3 (phospho S28) antibody [E191] (ChIP Grade) (ab303150)
- HRP Anti-Histone H3 (phospho S28) antibody [E191] (ChIP Grade) (ab303151)
- Alexa Fluor® 594 Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab310398)
- Alexa Fluor® 555 Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab311925)
- Alexa Fluor® 568 Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab312396)
- Anti-Histone H3 (phospho S28) antibody [E191] - ChIP Grade (ab32388)
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab215532 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 |
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ChIP |
Use at an assay dependent concentration.
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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Dot blot |
Use at an assay dependent concentration.
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ICC/IF |
Use at an assay dependent concentration.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 17 kDa.
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IP |
Use at an assay dependent concentration.
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Notes |
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ChIP
Use at an assay dependent concentration. |
Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Dot blot
Use at an assay dependent concentration. |
ICC/IF
Use at an assay dependent concentration. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 17 kDa. |
IP
Use at an assay dependent concentration. |
Target
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Function
Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. -
Sequence similarities
Belongs to the histone H3 family. -
Developmental stage
Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation. -
Post-translational
modificationsAcetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins. -
Cellular localization
Nucleus. Chromosome. - Information by UniProt
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Database links
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
see all -
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
see all
Images
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Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab32388 (red), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
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Western blot was performed on the immunoprecipitate using ab32388 at 1:500 dilution (2.444 μg/ml). VeriBlot for IP secondary antibody (HRP) (ab131366) at 1:1000 dilution.
Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
All lanes :
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 0.5µM Nocodazole for 24h whole cell lysate at 10 µg
Lane 2 : ab32388 IP in Nocodazole treated HeLa whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab32388 in nocodazole treated HeLa whole cell lysate
Observed band size: 17 kDa why is the actual band size different from the predicted? -
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, LP starved and non-starved, labeling anti-Histone H3 (phospho S28) with Ab32388 at 1/100 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor®488 (ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing nuclear staining on M phase of HeLa cells, then the signal decreased after LP treatment.
For the pan antibody, there was no great difference after LP treatment. The data showed mostly nuclear staining
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
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Dot blot performed using ab32388 at a dilution of 1/100. Lane 1 - Unmodified H3 peptide. Lane 2 - H3S28ph peptide. Lane 3 - H3.3S28ph peptide. Lane 4 - H3.3S31ph peptide. A HRP conjugated goat anti-rabbit (H+L) was used as the secondary antibody at a dilution of 1/2500. The exposure time was 3 minutes and the dilution and blocking buffer used were 5% NFDM/TBST.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
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IHC image of ab32388 staining Histone H3 in Human normal 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 ab32388, 0.1ug/ml, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
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Overlay histogram showing HeLa cells stained with ab32388 (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 (ab32388, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
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Overlay histogram showing HeLa cells stained with ab32388 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 90% methanol. 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 (ab32388, 1/1200 dilution, 1.02 µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730) (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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32388).
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (0)
ab215532 has not yet been referenced specifically in any publications.