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    products/cell-lysates/human-rara-retinoic-acid-receptor-alpha-knockout-hela-cell-lysate-ab257629.pdf

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Epigenetics and Nuclear Signaling Transcription Domain Families Zinc Finger
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Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell lysate (ab257629)

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Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell lysate (ab257629)
  • Sanger Sequencing - Human RARA knockout HeLa cell lysate (ab257629)

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Overview

  • Product name

    Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell lysate
    See all Retinoic Acid Receptor alpha kits
  • Product overview


    Knockout cell lysate achieved by CRISPR/Cas9.

  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon7.
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing
  • Reconstitution notes

    To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.

    *Usage of SDS sample buffer is not recommended with these lyophilized lysates.

  • Notes

    Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.

    User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

    Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
    See here for more information on knockout cell lysates.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

    This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

  • Tested applications

    Suitable for: WBmore details

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 1 kit
    ab262168 - Human RARA knockout HeLa cell lysate 1 x 100µg
    ab255929 - Human wild-type HeLa cell lysate 1 x 100µg
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • Zinc Finger
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • Nuclear Hormone Receptors
    • Retinoic & Retinoid
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • Nuclear Receptors
    • Retinoic & Retinoid
    • Cancer
    • Signal transduction
    • Nuclear signaling
    • Nuclear hormone receptors
    • Retanoic and retanoid
    • Neuroscience
    • Processes
  • Cell type

    epithelial
  • Disease

    Adenocarcinoma
  • Gender

    Female
  • STR Analysis

    Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10

Target

  • Function

    Receptor for retinoic acid. Retinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all-trans or 9-cis retinoic acid, and regulate gene expression in various biological processes. The RXR/RAR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. In the absence of ligand, the RXR-RAR heterodimers associate with a multiprotein complex containing transcription corepressors that induce histone acetylation, chromatin condensation and transcriptional suppression. On ligand binding, the corepressors dissociate from the receptors and associate with the coactivators leading to transcriptional activation. RARA plays an essential role in the regulation of retinoic acid-induced germ cell development during spermatogenesis. Has a role in the survival of early spermatocytes at the beginning prophase of meiosis. In Sertoli cells, may promote the survival and development of early meiotic prophase spermatocytes. In concert with RARG, required for skeletal growth, matrix homeostasis and growth plate function (By similarity). Regulates expression of target genes in a ligand-dependent manner by recruiting chromatin complexes containing MLL5. Mediates retinoic acid-induced granulopoiesis.
  • Involvement in disease

    Note=Chromosomal aberrations involving RARA are commonly found in acute promyelocytic leukemia. Translocation t(11;17)(q32;q21) with ZBTB16/PLZF; translocation t(15;17)(q21;q21) with PML; translocation t(5;17)(q32;q11) with NPM. The PML-RARA oncoprotein requires both the PML ring structure and coiled-coil domain for both interaction with UBE2I, nuclear microspeckle location and sumoylation. In addition, the coiled-coil domain functions in blocking RA-mediated transactivation and cell differentiation.
  • Sequence similarities

    Belongs to the nuclear hormone receptor family. NR1 subfamily.
    Contains 1 nuclear receptor DNA-binding domain.
  • Domain

    Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.
  • Post-translational
    modifications

    Phosphorylated on serine and threonine residues. Phosphorylation does not change during cell cycle. Phosphorylation on Ser-77 is crucial for transcriptional activity (By similarity). Phosphorylation by AKT1 is required for the repressor activity but has no effect on DNA binding, protein stability nor subcellular localization. Phosporylated by PKA in vitro. This phosphorylation on Ser-219 and Ser-369 is critical for ligand binding, nuclear localization and transcriptional activity in response to FSH signaling.
    Sumoylated by SUMO2, mainly on Lys-399 which is also required for SENP6 binding. On all-trans retinoic acid (ATRA) binding, a confromational change may occur that allows sumoylation on two additional site, Lys-166 and Lys-171. Probably desumoylated by SENP6. Sumoylation levels determine nuclear localization and regulate ATRA-mediated transcriptional activity.
    Trimethylation enhances heterodimerization with RXRA and positively modulates the transcriptional activation.
    Ubiquitinated.
  • Cellular localization

    Nucleus. Cytoplasm. Nuclear localization depends on ligand binding, phosphorylation and sumoylation. Transloaction to the nucleus in the absence of ligand is dependent on activation of PKC and the downstream MAPK phosphorylation.
  • Target information above from: UniProt accession P10276 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • NR1B1
    • Nuclear mitotic apparatus protein retinoic acid receptor alpha fusion protein
    • Nuclear receptor subfamily 1 group B member 1
    • Nucleophosmin retinoic acid receptor alpha fusion protein NPM RAR long form
    • RAR
    • RAR alpha
    • RAR-alpha
    • rara
    • RARA_HUMAN
    • RARalpha
    • RARalpha1
    • Retinoic acid nuclear receptor alpha variant 1
    • Retinoic acid nuclear receptor alpha variant 2
    • Retinoic acid receptor alpha
    • Retinoic acid receptor alpha polypeptide
    see all

Associated products

  • KO cell lines

    • Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell line (ab265176)
  • Related Products

    • Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] (ab275745)
    • Anti-Retinoic Acid Receptor alpha antibody [EPR23871-271] - BSA and Azide free (ab275748)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab257629 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
WB
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Images

  • Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell lysate (ab257629)
    Western blot - Human RARA (Retinoic Acid Receptor alpha) knockout HeLa cell lysate (ab257629)

    Lane 1: Wild-type HeLa cell lysate 20 ug
    Lane 2: RARA knockout HeLa cell lysate 20 ug
    Lane 3: MCF7 cell lysate 20 ug
    Lane 4: HepG2 cell lysate 20 ug

    Lanes 1 - 4: Merged signal (red and green). Green - ab275745 observed at 40 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.#ab275745 was shown to react with Retinoic Acid Receptor alpha in wild-type HeLa cells in western blot. The bands observed in RARA CRISPR-Cas9 edited cell line ab265176 (RARA CRISPR-Cas9 edited cell lysate ab257629) below 40 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and RARA CRISPR-Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab275745 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

  • Sanger Sequencing - Human RARA knockout HeLa cell lysate (ab257629)
    Sanger Sequencing - Human RARA knockout HeLa cell lysate (ab257629)
    Homozygous: 1 bp insertion in exon7

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 ab257629? Please let us know so that we can cite the reference in this datasheet.

ab257629 has not yet been referenced specifically in any publications.

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