AbFlex^® Histone H3.3 antibody (rAb)

Application Notes

Applications Validated by Active Motif:
ChIP-Seq: 4 µg per ChIP
WB*: 2 µg/ml dilution

*Note: many chromatin-bound proteins are not soluble in a low salt nuclear extract and fractionate to the pellet. Therefore, we recommend a High Salt / Sonication Protocol when preparing nuclear extracts for Western Blot.

Published Applications

The following applications have been published using this antibody. Unless noted above, Active Motif may not have validated the antibody for use in these applications:

CUT&RUN
ChIP-Seq
IF
WB

View publications that use Active Motif products & services here. Enter the product number(s) to see publications & applications that use this antibody.

Immunogen

This antibody was raised against a peptide corresponding to amino acids 21-39 of human Histone H3.3.

Buffer

Purified IgG in 140 mM Hepes, pH 7.5, 70 mM NaCl, 35 mM NaOAc, 0.035% sodium azide, 30% glycerol

Background

AbFlex^® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6xHis Tag, a Biotinylation Tag for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports.

AbFlex^® Histone H3.3 antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells.

Histone H3.1 and Histone H3.3 are the two main Histone H3 variants found in plants and animals. They are known to be important for gene regulation. Histone H3.1 and H3.3 have been shown to demonstrate unique genomic localization patterns thought to be associated with their specific functions in regulation of gene activity. Specifically, Histone H3.1 localization is found to coincide with genomic regions containing chromatin repressive marks (H3K9me3, H3K27me3 and DNA methylation), whereas Histone H3.3 primarily colocalizes with marks associated with gene activation (H3K4me3, H2BK120ub1, and RNA pol II occupancy). Deposition of the Histone H3.1 variant into the nucleosome correlates with the canonical DNA synthesis-dependent deposition pathway, whereas Histone H3.3 primarily serves as the replacement Histone H3 variant outside of S-phase, such as during gene transcription. Aberrant localization of these variants is also known to correlate with certain cancers.

Chromatin IP Assays

This antibody has been validated for use in ChIP and/or ChIP-Seq, and can be used with Active Motif's ChIP-IT^® High Sensitivity Kit or our magnetic bead-based ChIP-IT^® Express Kits. For an overview of all of our ChIP products, please visit our Chromatin IP Page to learn about ChIP products designed for use with AbFlex^® Histone H3.3 antibody (rAb).

Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20°C for up to 2 years. Keep all reagents on ice when not in storage.

Guarantee

This product is guaranteed for 12 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

• ChIP = Chromatin Immunoprecipitation;
• ChIP = ChIP Sequencing;
• CUT&RUN = Cleavage Under Targets and Release Using Nuclease;
• CUT&Tag = Cleavage Under Targets and Tagmentation;
• DB = Dot Blot;
• ELISA = Enzyme-linked Immunosorbent Assay;
• EMSA = Electrophoretic Mobility Shift Assay
• FC = Flow Cytometry;
• ICC = Immunocytochemistry;
• IF = Immunofluorescence;
• IHC = Immunohistochemistry;
• IP = Immunoprecipitation;
• MeDIP = Methyl-DNA Immunoprecipitation;
• NEU = Neutralizing;
• WB = Western Blotting