Anti-Orai1 (extracellular) Antibody

TMEM142A, CRACM1, Calcium release-activated calcium channel protein 1
    Cat #: ACC-062
  • Lyophilized Powder
  • Antigen Incl.
  • Shipped at Room Temp.
  • Type: Polyclonal
    Source: Rabbit
    Reactivity: m, r
    Immunogen
    Peptide (C)KFLPLKRQAGQPS, corresponding to amino acid residues 200-212 of mouse Orai1 (Accession Q8BWG9). 2nd extracellular loop.
    Accession (Uniprot) Number Q8BWG9
    Gene ID 109305
    Peptide confirmation Confirmed by amino acid analysis and mass spectrometry.
    Homology Rat - identical.
    Purity Affinity purified on immobilized antigen.
    Formulation Lyophilized powder. Reconstituted antibody contains phosphate buffered saline (PBS), pH 7.4, 1% BSA, 0.05% NaN3.
    Specificity Will not recognize human Orai1.
    Storage before reconstitution The antibody ships as a lyophilized powder at room temperature. Upon arrival, it should be stored at -20°C.
    Reconstitution 25 µl, 50 µl or 0.2 ml double distilled water (DDW), depending on the sample size.
    Antibody concentration after reconstitution 0.65 mg/ml.
    Storage after reconstitution The reconstituted solution can be stored at 4°C for up to 1 week. For longer periods, small aliquots should be stored at -20°C. Avoid multiple freezing and thawing. Centrifuge all antibody preparations before use (10000 x g 5 min).
    Control antigen storage before reconstitution Lyophilized powder can be stored intact at room temperature for 2 weeks. For longer periods, it should be stored at -20°C.
    Control antigen reconstitution 100 µl double distilled water (DDW).
    Control antigen storage after reconstitution -20°C.
    Preadsorption Control 1 µg peptide per 1 µg antibody.
    Standard quality control of each lot Western blot analysis.
    Applications: ic, ifc, ih, lci, wb
    May also work in: ip
    Western blot
    Western blot analysis of rat spleen (lanes 1 and4), rat pancreas (lanes 2 and 5) and mouse B-cell lymphoma (WEHI) (lanes 3 and 6) lysates:
    1-3. Anti-Orai1 (extracellular) Antibody (#ACC-062), (1:200).
    4-6. Anti-Orai1 (extracellular) Antibody, preincubated with the control peptide antigen.
    Immunohistochemistry
    Expression of Orai1 in rat pancreas
    Immunohistochemical staining of rat pancreas paraffin embedded sections using Anti-Orai1 (extracellular) Antibody (#ACC-062), (1:50). Orai1 (brown) is expressed in acinar cells. Hematoxilin is used as the counterstain.
    Indirect flow cytometry
    Indirect flow cytometry analysis of WEHI living cells:
    ___ Unstained cells.
    ___ Cells + Anti-Orai1 (extracellular) Antibody (#ACC-062), (10 µg/0.5 x 106 cells).
    The control antigen is not suitable for this application.
    Live cell imaging / Immunocytochemistry
    Expression of Orai1 in rat RBL cells
    Immunocytochemical staining of intact living rat basophilic leukemia (RBL) cells. A. Extracellular staining of cells using Anti-Orai1 (extracellular) Antibody (#ACC-062), (1:100) followed by goat anti-rabbit-AlexaFluor-488 secondary antibody (green). B. Merge of A and Live view of the cells.
    References
    1. Eisner, D.A. et al. (2005) Exp. Physiol. 90, 3.
    2. Chakrabarti, R. and Chakrabarti, R. (2006) J. Cell. Biochem99, 1503.
    3. Feske, S. et al. (2006) Nature 441, 179.
    4. Pickett, J. (2006) Nature Reviews Mol. Cell Biol. 7, 794.
    5. Luik, R.M. et al. (2006) J. Cell. Biol174, 815.
    6. Wu, M.M. et al. (2006) J. Cell. Biol174, 803.
    7. Ong, H.L. et al. (2007) J. Biol. Chem. 282, 9105.
    8. Luik, R.M and Lewis, R.S. (2007) Trends Mol. Med. 13, 103.
    9. DeHaven, W.I. et al. (2007) J. Biol. Chem. 282, 17548.
    Scientific background

    Cytosolic calcium (Ca2+) has long been known to act as a key second messenger in many intracellular pathways including synaptic transmission, muscle contraction, hormonal secretion, cell growth and proliferation1,2. Intracellular Ca2+ levels are controlled by either the influx of Ca2+ through the calcium-release-activated Ca2+ channels (CRAC), or from intracellular stores which gained much attention.

    Recently, several key players of the store operated complex have been identified3Orai1 (also known as CRACM1) acts as the store operated Ca2+ channel (SOC) and STIM1, which acts as the endoplasmic reticulum Ca2+ sensor3,4. The formation of functional channels requires the presence of both Orai1 and STIM1 proteins working as a complex and involves the co-clustering of Orai1 on the plasma membrane with STIM1 on the endoplasmic reticulum4-6TRPC1, a member of the transient receptor potential family was also suggested to act as a player in the SOC complex7. In T-cells, Ca2+ entry following activation by antigen-receptor engagement occurs solely through CRAC channels where Orai1 constitutes the pore forming subunit3,8.

    Orai1 is a plasma membrane protein with four potential transmembrane domains and intracellular N- and C-terminus. In addition, two mammalian homologs to Orai1 have been identified; Orai2 and Orai33,9. All three, Orai1 Orai2 and Orai3, are capable of forming store operated channels with different magnitudes9.

    Application key:

    CBE- Cell-based ELISA, FC- Flow cytometry, ICC- Immunocytochemistry, IE- Indirect ELISA, IFC- Indirect flow cytometry, IHC- Immunohistochemistry, IP- Immunoprecipitation, LCI- Live cell imaging, N- Neutralization, WB- Western blot

    Species reactivity key:

    H- Human, M- Mouse, R- Rat
    Image & Title: Anti-Orai1 (extracellular) Antibody
    Expression of Orai1 in mouse optic nerve.A(i-iv). Immunohistochemical staining of mouse optic nerve sections using Anti-Orai1 (extracellular) Antibody (#ACC-062). Orai1 staining (green) is detected in astrocytes and co-localizes with GFAP staining (red) an astrocyte marker. A(v). Immunocytochemical staining of mouse cultured optic nerve cells using Anti-Orai1 (extracellular) Antibody, (green). Note the lack of immunostaining when the antibody is pre-incubated with the control antigen (Aiii, inset).Adapted from Papanikolaou, M. et al. (2017) Brain Struct. Funct. 222, 2993. with permission of SPRINGER NATURE.
    Last update: 22/11/2018

    Anti-Orai1 (extracellular) Antibody (#ACC-062) is a highly specific antibody directed against an extracellular epitope of the mouse protein. The antibody can be used in western blot, immunohistochemistry, live cell imaging, and indirect flow cytometry applications. It has been designed to recognize the Orai1 channel from mouse and rat samples.

    For research purposes only, not for human use
    Citations
    Western blot citations
    1. Rat pulmonary arterial endothelial cell lysate (1:400).
      Zhang, B. et al. (2018) Am. J. Physiol. 314, H359.
    2. Mouse C2C12 myoblast cell lysate.
      Garcia-Castaneda, M. et al. (2017) J. Physiol. 595, 4167.
    3. Mouse renal artery lysate.
      Kuang, S.J. et al. (2017) Eur. J. Pharmacol. 805, 93.
    4. Rat lung lysate (1:500).
      Zhang, W. et al. (2017) Inflammation 40, 778.
    5. Mouse pulmonary arterial smooth muscle cells.
      Gonzalez Bosc, L.V. et al. (2016) Am. J. Physiol. 311, L48.
    6. Mouse splenocyte lysate (1:500).
      Lisak, D. et al. (2016) Cell Death Differ. 23, 358.
    7. Mouse HT22R cell lysate.
      Henke, N. et al. (2013) Cell Death Dis. 4, e470.
    Immunohistochemistry citations
    1. Mouse optic nerve sections.
      Papanikolaou, M. et al. (2017) Brain Struct. Funct. 222, 2993.
    2. Rat lung sections (1:100).
      Zhang, W. et al. (2017) Inflammation in 40, 778.
    3. Rat spinal cord sections.
      Qi, Z. et al. (2016) Cell Mol. Neurobiol. 36, 1035.
    Immunocytochemistry citations
    1. Mouse RAW264.7 macrophage-like cells.
      Li, H. et al. (2017) Sci. Rep. 7, 226.
    2. Mouse optic nerve cell.
      Papanikolaou, M. et al. (2017) Brain Struct. Funct. 222, 2993.
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