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    chiccutrun-pag-mnase-ab285373.pdf

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ChIC/CUT&RUN pAG-MNase (ab285373)

  • Datasheet
  • SDS
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SDS-PAGE - ChIC/CUT&RUN pAG-MNase (ab285373)
  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)
  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)
  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)

Key features and details

  • Expression system: Escherichia coli
  • Active: Yes
  • Suitable for: ChIC/CUT&RUN, ChIC/CUT&RUN-seq

Description

  • Product name

    ChIC/CUT&RUN pAG-MNase
  • Biological activity

    pAG-MNase enzyme for performing ChIC/CUT&RUN experiments. Biological activity determined by using pAG-MNase in the ChIC/CUT&RUN experiment on 1 X105 HeLa cells with ab188408 Anti-CTCF antibody followed by sequencing. Click here for the full list of ChIC/CUT&RUN-seq validated antibodies.

    pAG-MNase is supplied in 10 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 50% glycerol. Use 2 µl pAG-MNase per 50 µl ChIC/CUT&RUN reaction (25X dilution). 

    Number of reactions:

    100 µl vial (50 reactions)

    500 µl vial (250 reactions)

    1. Schmid M, Durussel T, Laemmli U K. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell, 16(1):147–57 (2004)

    2. Skene P J, Henikoff J G, Henikoff S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc, 13:1006–1019 (2018)

  • Expression system

    Escherichia coli
  • Protein length

    Full length protein
  • Animal free

    No
  • Nature

    Recombinant
    • Additional sequence information

      Nuclease
  • Description

    Recombinant MNase protein

Specifications

Our Abpromise guarantee covers the use of ab285373 in the following tested applications.

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

  • Applications

    ChIC/CUT&RUN

    ChIC/CUT&RUN sequencing

  • Form

    Liquid
  • Additional notes

    ChIC was first described by the Laemmli group in 2004 (1). The ChIC method involves cleavage of double-stranded DNA (1). The antibody directed pAG-MNAse fusion protein (ab285373) results in 100 – 200 bp resolution mapping of DNA-bound proteins (1, 2). ChIC/CUT&RUN-seq allows chromatin immunoprecipitation to be performed on fewer than 100 cells for histone modifications and ~1000 cells for transcription factors compared to ChIP-seq which generally needs >106 cells (1, 3). Another advantage of ChIC/CUT&RUN-seq over ChIP-seq is an ~10-fold lower sequencing depth (1, 3). Abcam provides an increasing number of ChIC/CUT&RUN-seq validated antibodies to meet the needs of this emerging technique.

    1. Schmid M, Durussel T, Laemmli U K. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell, 16(1):147–57 (2004)

    2. Skene P J, Henikoff J G, Henikoff S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc, 13:1006–1019 (2018)

    3. Skene P J, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife, e21856 (2017)

  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.

    Information available upon request.

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

Images

  • SDS-PAGE - ChIC/CUT&RUN pAG-MNase (ab285373)
    SDS-PAGE - ChIC/CUT&RUN pAG-MNase (ab285373)

    Figure 1: Protein gel of pAG-MNAse

    pAG-MNase ab285373 (16 µg per lane) was analysed by SDS-PAGE and stained using Coomassie Blue. The molecular weights are outlined alongside the target protein.

  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)
    Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)

    Figure 2: Size distribution of cleaved chromatin

    ChIC/CUT&RUN was performed with ab285373 pAG-MNase (1:25 dilution) and Anti-CTCF antibody (ab188408). Library preparation was performed and the size of excised DNA fragments was analysed by an Agilent 4200 TapeStation. Enriched cleaved DNA mononucleosomes are observed at ~300 bp.

  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)
    Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)

    Figure 3: ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408)

    ChIC/CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 1x105 HeLa cells and 5µg of ab188408 [EPR18253]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 1x107 HeLa cells and 4 µg of ab188408 [EPR18253]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

  • Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)
    Functional Studies - ChIC/CUT&RUN pAG-MNase (ab285373)

    Figure 4: ChIC/CUT&RUN sequencing - Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade (ab213224)

    ChIC/CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 1x105 HeLa cells and 2µg of ab213224 [EPR20551-225]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 1x107 HeLa cells and 4 µg of ab213224 [EPR20551-225]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

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.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab285373? Please let us know so that we can cite the reference in this datasheet.

ab285373 has not yet been referenced specifically in any publications.

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