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ORMDL3 anticorps (N-Term)

ORMDL3 Reactivité: Humain, Souris, Rat, Cobaye, Cheval, Lapin, Poisson zèbre (Danio rerio), Boeuf (Vache), Chien, Chévre WB Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN2774396
  • Antigène Voir toutes ORMDL3 Anticorps
    ORMDL3 (ORM1-Like 3 (ORMDL3))
    Épitope
    • 8
    • 8
    • 6
    • 6
    • 5
    • 1
    • 1
    • 1
    N-Term
    Reactivité
    • 31
    • 13
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Humain, Souris, Rat, Cobaye, Cheval, Lapin, Poisson zèbre (Danio rerio), Boeuf (Vache), Chien, Chévre
    Hôte
    • 31
    Lapin
    Clonalité
    • 31
    Polyclonal
    Conjugué
    • 11
    • 4
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Cet anticorp ORMDL3 est non-conjugé
    Application
    • 19
    • 17
    • 2
    • 2
    • 1
    • 1
    Western Blotting (WB)
    Séquence
    GTAHSEVNPN TRVMNSRGIW LSYVLAIGLL HIVLLSIPFV SVPVVWTLTN
    Specificité
    Pan specific ORMDL1-3 antibody. Predicted to react with ORMDL1, ORMDL2 and ORMDL3.
    Homologie
    Cow: 100%, Dog: 100%, Goat: 100%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%, Zebrafish: 100%
    Attributs du produit
    This is a rabbit polyclonal antibody against ORMDL3. It was validated on Western Blot.
    Purification
    Affinity Purified
    Top Product
    Discover our top product ORMDL3 Anticorps primaire
  • Indications d'application
    Optimal working dilutions should be determined experimentally by the investigator.
    Commentaires

    Antigen size: 153 AA

    Restrictions
    For Research Use only
  • Validation #103540 (Western Blotting)
    'Independent Validation' signe
    by
    Sita Devi Gupta Ph.D., Dr Teresa M Dunn Lab, Uniformed Services University of the Health Sciences
    No.
    #103540
    Date
    03.12.2018
    Antigène
    ORMDL3, 1, 2
    Numéro du lot
    68904-43140
    Application validée
    Western Blotting
    Contrôle positif

    Jurkat and HEK-293 cells for endogenous protein

    Flp-In T-Rex HEK-293 cells expressing Fusion SPT on induction with Tetracycline, which in turn induces endogenous ORMDLs. (https://www.ncbi.nlm.nih.gov/pubmed/25395622)

    Transient expression of NubG-HA ORMDL1, 2, or 3 in (PCR cloned from human gene expression library into yeast vector) HEK-293 cells

    Transient expression of ORMDL1-GFP in HEK-293 cells

    Jurkat cells stably expressing ORMDL3-HA generated using pUNO-ORMDL3-HA construct and blasticidine selection

    Purified ORMDL1/2/3-GFP

    Contrôle négative

    siRNA against all the ORMDLs transfected together in Flp-In T-Rex HEK293 cells harboring Tet inducible Fusion serine palmitoyl transferase (LCB2a-ssSPTa-LCB1) SPT without and with Tet. siRNA from Dharmacon each one as ON-TARGET plus Human ORMDL1 (94101), human ORMDL2 (29095) and human ORMDL3 (94193) siRNA-SMARTpool.

    Conclusion

    Passed. ABIN2774396 specifically recognizes endogenous ORMDLs, transfected ORMDL1, 2, and 3 in HEK 293 cell lysates and purified ORMD1/2/3-GFP fusion proteins.

    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN2774396
    Anticorps secondaire
    goat anti-Rabbit IgG HRP conjugate (Bio-Rad, 170-6515, lot L005679 A)
    Full Protocol
    • 293T cells:
      • Grow 293T (ATCC) or Flp-In T-Rex HEK-293 (Invitrogen) cells in DMEM (ThermoFisher Scientifc, 10569044, lot 1929910) supplemented with 10% FBS (JRH Biosciences, 12103-78P, lot 4M2224) and penicillin-streptomycin (ThermoFisher Scientifc, 15140122, lot 1864852) at 37°C and 5% CO2 in 2ml on a 6 well dish to 60% confluency.
      • Transfect 293T cells with 750ng NubG-HA-ORMDL1, 2, or 3 in pcDNA or ORMDL1-GFP fusion protein cloned into in pCMV6-ACGFP (Origene, RG202670) along with human LCB1, 2a and NubG-HA-ssSPTa using lipofectamine 2000 (ThermoFisher Scientific, 11668019 , lot 1959316 or 1992885) following the manufacturer´s instructions.
      • Grow cells for 36h at 37°C and 5% CO2.
    • Jurkat cells:
      • Grow Jurkat Clone E6 (ATCC, TIB-152) and Jurkat cells stably expressing ORMDL3-HA RPMI-1640 Medium (ATCC,30-2001) supplemented with 10% FBS (JRH Biosciences, 12103-78P, lot 4M2224) at 37°C and 5% CO2 in 2ml on a 6 well dish to 60% confluency.
    • Flp-In T-Rex HEK-293:
      • Grow Flp-In T-Rex HEK-293 (Invitrogen) with tetracycline inducible fusion-SPT cells in DMEM (ThermoFisher Scientifc, 10569044, lot 1929910) supplemented with 10% FBS (JRH Biosciences, 12103-78P, lot 4M2224) and penicillin-streptomycin (ThermoFisher Scientifc, 15140122, lot 1864852) at 37°C and 5% CO2 in 2ml on a 6 well dish to 60% confluency.
      • For tet-induction of SPT expression add 2µg/ml tetracyline.
      • For ORMDLs siRNA knockdown transfect a mix of 0.8ml optiMEM (each siRNA 7.5µl from 20µM stock diluted in 0.4ml optiMEM + 15µl lipofectamine in 0.4ml optiMEM) with a final concentration of50nM of ORMDL1 (Dharmacon, 94101), ORMDL2 (Dharmacon, 29095), and ORMDL3 (Dharmacon, 94193) siRNA each into Flp-In T-Rex HEK293 cells (2.2ml optiMEM) harboring tet-inducible Fusion serine palmitoyl transferase (LCB2a-ssSPTa-LCB1) SPT using lipofectamine 2000 (ThermoFisher Scientific, 11668019, lot 1959316 or 1992885) following the manufacturer´s instructions into a final volume of 3ml. After 10h incubation, add complete DMEM media with 2µg/ml tetracycline and incubate for 46h.
    • Wash cells twice with ice-cold PBS.
    • Lyse 1x106 cells in 200-250µl per well cold lysis buffer: 2 volumes RIPA buffer (50mM Tris-HCl pH8, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and 1mM EDTA) and 1 volume membrane buffer (50mM Tris-HCl pH7.5, 1mM EGTA, 30 % glycerol), protease and phosphatase inhibitors.
    • Determine total protein content of the lysates using Protein Assay Dye (Bio-Rad, 5000006, lot L007096 C).
    • Denature about 200µg (35µg protein for Flp-In T-Rex HEK293 cells) of total protein for 14min at 70°C in 100µl NuPAGE LDS sample buffer (ThermoFisher Scientific, NP0008, lot 736752) and subsequently separate them on a 4-12% Bis-Tris Protein Gel (ThermoFisher Scientific, NP0336BOX, lot 18092071) at 150V for 10min and 185V for 40min.
    • Transfer proteins onto nitrocellulose membrane (Bio-Rad, 1620213, lot 1603053) for 2h at 85V.
    • Block membrane with TBST (20mM Tris-HCl, pH7.5, 150mM NaCl with 0.1% Tween 20) and 5% Blocking-Grade Blocker (Bio-Rad, 170-6404, lot L005668 A) for 1h at RT.
    • Incubation with rabbit anti-ORMDL3 (antibodies-online, ABIN2774396, lot 68904-43140) diluted 1:1000 in TBST with 2% Blocking-Grade Blocker for 1h at RT.
    • Wash membrane with deionized water.
    • Wash membrane 3x for 10min with TBST.
    • Incubation with secondary goat anti-Rabbit IgG HRP conjugate (Bio-Rad, 170-6515, lot L005679 A) diluted 1:5000 in TBST with 2% Blocking-Grade Blocker for 1h at RT.
    • Wash membrane with deionized water and then 4x for 10min with TBST.
    • Reveal protein bands using Western Lightning Plus-ECL (PerkinElmer, NEL105001EA, lot 203-17431) on a Kodak X-OMAT 2000A processor using Radiomat LS 7980052 films (MidSci).
    Notes
    • ABIN2774396 reveals a protein of the expected molecular weight in lysates of 293T and in Flp-In T-Rex HEK293 cells harboring Tet inducible Fusion SPT upon Tet induction. It also showed the expected size protein band in HEK-293 cells transiently transfected with ORMDL1/2 or 3 tagged recombinant plasmids and with the purified ORMDL-GFPs (GFP fusion proteins run higher due to the tag).

    • ABIN2774396 did not detect recombinant ORMDL3-HA in total lysates from Jurkat cells stably expressing ORMDL3-HA.

    • ORMDL protein bands are visible in the positive samples and were significantly decreased upon knockdown using ORMDLs siRNAs. siRNA knockdown showed reduced expression of ORMDLs in uninduced and Tet induced fusion SPT cells as compared to the respective controls.

    • In Jurkat cells the signal is very week but longer exposure reveals the protein at the expected molecular weight. With 293T cells we see a stronger band just above 21kDa. This band was absent in Jurkat cells and in the purified proteins.

  • Format
    Liquid
    Concentration
    Lot specific
    Buffer
    Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.
    Agent conservateur
    Sodium azide
    Précaution d'utilisation
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Conseil sur la manipulation
    Avoid repeated freeze-thaw cycles.
    Stock
    -20 °C
    Stockage commentaire
    For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.
  • Antigène
    ORMDL3 (ORM1-Like 3 (ORMDL3))
    Autre désignation
    ORMDL3 (ORMDL3 Produits)
    Synonymes
    anticorps 2810011N17Rik, anticorps AI315223, anticorps LRRGT00183, anticorps ORMDL sphingolipid biosynthesis regulator 3, anticorps ORM1-like 3 (S. cerevisiae), anticorps ORMDL3, anticorps Ormdl3
    Sujet
    ORMDL3 is a negative regulator of sphingolipid synthesis. ORMDL3 may indirectly regulate endoplasmic reticulum-mediated Ca(+2) signaling.
    Alias Symbols: -
    Protein Interaction Partner: UBC, APP, LNX1, ELAVL1, SPTLC1, EEF1A1,
    Protein Size: 153
    Poids moléculaire
    17 kDa
    ID gène
    94103
    NCBI Accession
    NM_139280, NP_644809
    UniProt
    Q8N138
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