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GLI2 antibody (GLI Family Zinc Finger 2) (Middle Region) Primary Antibody

GLI2 Reactivity: Human IHC, WB Host: Rabbit Polyclonal unconjugated
Pubmed (23) Independent Validation (2)
Catalog No. ABIN2777474
$339.00
Plus shipping costs $45.00
100 μL ABIN2777474
100 μL ABIN2777474
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  • Target
    GLI2
    Binding Specificity
    • 10
    • 8
    • 8
    • 3
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    Middle Region
    Reactivity
    • 46
    • 14
    • 6
    • 2
    • 2
    • 1
    Human
    Host
    • 39
    • 7
    • 3
    Rabbit
    Clonality
    • 46
    • 3
    Polyclonal
    Conjugate
    • 22
    • 4
    • 3
    • 3
    • 3
    • 3
    • 3
    • 3
    • 1
    • 1
    • 1
    • 1
    • 1
    This GLI2 antibody is un-conjugated
    Application
    • 26
    • 21
    • 13
    • 6
    • 6
    • 5
    Immunohistochemistry (IHC), Western Blotting (WB)
    Sequence
    RNDVHLRTPL LKENGDSEAG TEPGGPESTE ASSTSQAVED CLHVRAIKTE
    Predicted Reactivity
    Human: 100%
    Characteristics
    This is a rabbit polyclonal antibody against GLI2. It was validated on Western Blot and immunohistochemistry.
    Purification
    Protein A purified
    Immunogen
    The immunogen is a synthetic peptide directed towards the N terminal region of human GLI2
  • Application Notes
    Optimal working dilutions should be determined experimentally by the investigator.
    Comment

    Antigen size: 1258 AA

    Restrictions
    For Research Use only
  • Validation #101239 (Western Blotting)
    'Independent Validation' Badge
    by
    Laboratory of Herditary Cancer, Division of Molecular Medicine, Rudjer Boskovic Institute
    No.
    #101239
    Date
    10/05/2017
    Antigen
    GLI2
    Lot Number
    QC49260-42551
    Method validated
    Western Blotting
    Positive Control

    Human melanoma cell line WM793B

    Human metastatic melanoma tissue

    Negative Control
    Human healthy skin not expressing GLI2
    Notes
    Passed. ABIN2777474 specifically recognizes GLI2 in WM793B cells and human metastatic melanoma tissue.
    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN2777474
    Secondary Antibody
    anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, 7074)
    Full Protocol
    • Grow WM793B cells in RPMI (Lonza, BE12-702F) supplemented with FBS (Sigma-Aldrich, F7524) and penicillin/streptomycin (Thermo Fisher Scientific, 15070063), at 37°C and 5% CO2 in a 10 cm TC-dish.
    • Wash cells 2x with PBS.
    • Harvest cells.
    • Lyse cells in 85µl cold lysis buffer (25mM Tris-HCl pH7.5, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with sonication 2x 15s using an Labsonic M sonicator with a 1mm probe (B. Braun Biotech International, 8535027), 1 cycle, 80% amplitude.
    • Determine total protein content of the lysates using a BCA protein assay (Thermo Fisher Scientific, 23225).
    • Denature 50μg of total proteins for 5min at 95°C in Laemmli SDS sample buffer and subsequently separate them on a freshly cast denaturing 7% PAA gel (using components from Sigma-Aldrich) for 2h at 100V.
    • Transfer proteins onto Amersham Protran Premium 0.2 NC membrane (GE Healthcare, 10600004) with a Mini Trans-Blot cell (Bio-Rad, 170-3930) for 80min at 100V.
    • Block the membrane with blocking buffer and blocking reagent (TBST/5% non-fat milk) for 30min at RT.
    • Incubation with primary rabbit anti human GLI2 antibody (antibodies-online, ABIN2777474, lot QC492960-42551) diluted 1:1000 in TBST/5% non-fat milk ON at 4°C.
    • Wash membrane 3x for 5min with TBST.
    • Incubation with secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, 7074) diluted 1:3000 in 5% non-fat milk/TBST for 1h at RT.
    • Wash membrane 3x for 5mins with TBST.
    • Reveal protein bands using Super Signal West Pico and Super Signal West Femto reagents (Thermo Fisher Scientific) on an UVITEC Alliance Imaging system (Cambridge).
    Experimental Notes
    • The GLI2 antibody ABIN2777474 reveals two protein bands of the expected molecular weight for the full-length (GLI2 FL) and the GLI2 N-terminally truncated (GLI2ΔN) isoform in lysates of WM793B cells.

    • ABIN2777474 also detects both GLI2 FL as well as GLI2ΔN in metastatic melanoma cell lysates whereas neither protein is revealed in human healthy skin tissue in which expression of GLI2 is not to be expected.

    • The predicted molecular weight of the full length GLI2 is 197kDa which is in accordance with our observation. With our previous anti-GLI2 antibody (sc-20291 (G-20), Santa Cruz Biotechnology) we also detected the GLI2ΔN protein at ~150kDa. The difference in apparent MW could be due to different migration of the marker.

  • Validation #102749 (Immunohistochemistry)
    'Independent Validation' Badge
    by
    Laboratory for Hereditary Cancer, Division of Molecular Medicine, Rudjer Boskovic Institute
    No.
    #102749
    Date
    02/02/2018
    Antigen
    GLI2
    Lot Number
    QC49260-42551
    Method validated
    Immunohistochemistry
    Positive Control
    Human colon cancer tissue sections
    Negative Control
    no primary antibody
    Notes

    Passed. ABIN2777474 specifically labels the targeted antigen in human colon cancer samples in IHC.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN2777474
    Secondary Antibody
    biotinylated anti-rabbit, streptavidin-HRP (LSAB2 System-HRP, Dako, K0675, lot 10126805)
    Full Protocol
    • Fix human colon cancer tissue in 10% buffered formalin, ON at RT.
    • Process tissue using an automated tissue processor (Leica or Tissue-Tec VIP Sakura):
      • 70% ethanol 2x for 30min.
      • 96% ethanol 2x for 30min.
      • Absolute ethanol 2x 30min.
      • Xylene substitute (Tissue-Clear, Sakura) 2x 30min.
      • Paraffin 2x 30min at 62°C. Embed tissue in paraffin blocks using the Tissue-Tec TEC embedding console system.
    • Cut paraffin embedded tissue with a Sliding microtome Slide 4003 E (PFM Medical) into 3µm sections.
    • Deparaffinize and rehydrate sections through a graded xylene and graded alcohol series:
      • Bioclear New xylene substitute (Biognost, BCN-1L, lot BCN-03/17) 3x for 3min.
      • 100% ethanol 2x for 1min.
      • 95% ethanol 2x for 1min.
      • 70% ethanol 2x for 1min.
      • H2O for 5min.
    • Incubate the sections in preheated citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH6.0) preheated to 100°C for 20min.
    • Cool sections to RT for 20min.
    • Rinse sections 1x for 1min with 1x TBST.
    • Block endogenus peroxidase by incubating the sections in 3% H2O2 in methanol.
    • Rinse sections 3x for 1min with 1x TBST.
    • Block sections in Protein Block (Dako, X0909, lot 10070336) for 20min at RT.
    • Blot excess serum from sections.
    • Incubate sections with primary rabbit anti-human GLI2 antibody (antibodies-online, ABIN2777474, lot QC492960-42551) diluted 1:100 in 2% BSA/TBST ON at 4°C. Include a no primary antibody negative control.
    • Wash sections 3x with TBST for 1min.
    • Incubate sections with biotinylated anti-rabbit and anti-mouse immunoglobulin (LSAB2 System-HRP, Dako, K0675, lot 10126805) for 10min at RT.
    • Wash sections 3x for 1min with TBST.
    • Incubate sections with streptavidin-HRP (LSAB2 System-HRP, Dako, K0675, lot 10126805) for 10min at RT.
    • Staining procedure with DAB chromogen (Dako, Liquid DAB+ Substrate Chromogen System, K3468, lot 10131855) and counterstaining with haematoxylin G2 (Biognost, HEMG2-OT-500, lot HEMG2-10/17).
    • Rinse sections 1x for 5min with distilled water.
    • Dehydrate in fresh ethanol:
      • 70% ethanol 2x for 1min.
      • 95% ethanol 2x for 1min.
      • 100% ethanol 2x for 1min.
      • Bioclear New (xylene substitute) 3x for 3min.
    • Mount sections in Biomount New (Biognost, BMN-30, lot BMN-10/16) mounting medium.
    • Dry sections in a ventilated place.
    • Image acquisition onthe Olympus BX51 microscope at 200x magnification.
    Experimental Notes
    • ABIN2777474 did not work with a protocol where the sections were incubated with epitope retrieval buffer at 85°C for 10min, and where the washing steps were done with PBS.

    • Localization of GLI2 is cytoplasmic and nuclear. No signal was detected in the secondary antibody only control.

  • Format
    Liquid
    Concentration
    Lot specific
    Buffer
    Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handling Advice
    Avoid repeated freeze-thaw cycles.
    Storage
    -20 °C
    Storage Comment
    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.
  • Bosco-Clément, Zhang, Chen, Zhou, Li, Mikami, Hirata, Yagui-Beltran, Lui, Do, Cheng, Tseng, Choi, Fang, Kim, Yue, Wang, Zheng, Fujii, Mann, Jablons, He: "Targeting Gli transcription activation by small molecule suppresses tumor growth." in: Oncogene, Vol. 33, Issue 16, pp. 2087-97, 2014 (PubMed).

    Li, Drnevich, Akraiko, Band, Li, Wang, Matoba, Tanaka: "Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells." in: Genomics, Vol. 102, Issue 5-6, pp. 456-67, 2013 (PubMed).

    Tostar, Finta, Rahman, Shimokawa, Zaphiropoulos: "Novel mechanism of action on Hedgehog signaling by a suppressor of fused carboxy terminal variant." in: PLoS ONE, Vol. 7, Issue 5, pp. e37761, 2012 (PubMed).

    Yang, Wang, Chen, Gatalica, Qiu, Liu, Stoner, Zhang, Weiss, Xie: "Hedgehog signaling activation in the development of squamous cell carcinoma and adenocarcinoma of esophagus." in: International journal of biochemistry and molecular biology, Vol. 3, Issue 1, pp. 46-57, 2012 (PubMed).

    Malek, Matta, Taylor, Perry, Mendrysa: "The p53 inhibitor MDM2 facilitates Sonic Hedgehog-mediated tumorigenesis and influences cerebellar foliation." in: PLoS ONE, Vol. 6, Issue 3, pp. e17884, 2011 (PubMed).

    Jung, Jung, Park, Song, Park: "Aberrant Hedgehog ligands induce progressive pancreatic fibrosis by paracrine activation of myofibroblasts and ductular cells in transgenic zebrafish." in: PLoS ONE, Vol. 6, Issue 12, pp. e27941, 2011 (PubMed).

    Schneider, Thurnher, Kloimstein, Leitner, Petzelbauer, Pammer, Brunner, Erovic: "Expression of the Sonic hedgehog pathway in squamous cell carcinoma of the skin and the mucosa of the head and neck." in: Head & neck, Vol. 33, Issue 2, pp. 244-50, 2011 (PubMed).

    Nakamura, Sasajima, Mizukami, Sugiyama, Yamazaki, Fujii, Kawamoto, Koizumi, Sato, Fujiya, Sasaki, Tanno, Okumura, Shimizu, Kawabe, Karasaki, Kono, Ii, Bardeesy, Chung, Kohgo: "Hedgehog promotes neovascularization in pancreatic cancers by regulating Ang-1 and IGF-1 expression in bone-marrow derived pro-angiogenic cells." in: PLoS ONE, Vol. 5, Issue 1, pp. e8824, 2010 (PubMed).

    Youssef, Van Keymeulen, Lapouge, Beck, Michaux, Achouri, Sotiropoulou, Blanpain: "Identification of the cell lineage at the origin of basal cell carcinoma." in: Nature cell biology, Vol. 12, Issue 3, pp. 299-305, 2010 (PubMed).

    Peacock, Cox, Schmidt: "Involvement of PTCH1 mutations in the calcifying epithelial odontogenic tumor." in: Oral oncology, Vol. 46, Issue 5, pp. 387-92, 2010 (PubMed).

    Coon, Laukert, Pedone, Laterra, Kim, Fults: "Molecular therapy targeting Sonic hedgehog and hepatocyte growth factor signaling in a mouse model of medulloblastoma." in: Molecular cancer therapeutics, Vol. 9, Issue 9, pp. 2627-36, 2010 (PubMed).

    Wall, Mears, McNeill, Mazerolle, Thurig, Wang, Kageyama, Wallace: "Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity." in: The Journal of cell biology, Vol. 184, Issue 1, pp. 101-12, 2009 (PubMed).

    Madison, McKenna, Dolson, Epstein, Kaestner: "FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine." in: The Journal of biological chemistry, Vol. 284, Issue 9, pp. 5936-44, 2009 (PubMed).

    Takanaga, Tsuchida-Straeten, Nishide, Watanabe, Aburatani, Kondo: "Gli2 is a novel regulator of sox2 expression in telencephalic neuroepithelial cells." in: Stem cells (Dayton, Ohio), Vol. 27, Issue 1, pp. 165-74, 2009 (PubMed).

    Kim, Singh, Cho-Vega, Drakos, Davuluri, Khokhar, Fayad, Medeiros, Vega: "Sonic hedgehog signaling proteins and ATP-binding cassette G2 are aberrantly expressed in diffuse large B-cell lymphoma." in: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, Vol. 22, Issue 10, pp. 1312-20, 2009 (PubMed).

    Kim, Wang, Wu, Doble, Patel, Woodgett, Snider: "GSK-3 is a master regulator of neural progenitor homeostasis." in: Nature neuroscience, Vol. 12, Issue 11, pp. 1390-7, 2009 (PubMed).

    Syn, Witek, Curbishley, Jung, Choi, Enrich, Omenetti, Agboola, Fearing, Tilg, Adams, Diehl: "Role for hedgehog pathway in regulating growth and function of invariant NKT cells." in: European journal of immunology, Vol. 39, Issue 7, pp. 1879-92, 2009 (PubMed).

    Ho, Stojanovski, Whetstone, Wei, Mau, Wunder, Alman: "Gli2 and p53 cooperate to regulate IGFBP-3- mediated chondrocyte apoptosis in the progression from benign to malignant cartilage tumors." in: Cancer cell, Vol. 16, Issue 2, pp. 126-36, 2009 (PubMed).

    Kiprilov, Awan, Desprat, Velho, Clement, Byskov, Andersen, Satir, Bouhassira, Christensen, Hirsch: "Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery." in: The Journal of cell biology, Vol. 180, Issue 5, pp. 897-904, 2008 (PubMed).

    Kabasawa, Nagumo, Takeda, Kawashima, Okada, Omura, Yamaguchi, Katsube: "Amelogenin positive cells scattered in the interstitial component of odontogenic fibromas." in: Journal of clinical pathology, Vol. 61, Issue 7, pp. 851-5, 2008 (PubMed).

  • Target
    GLI2
    Alternative Name
    GLI2 (GLI2 Antibody Abstract)
    Synonyms
    HPE9, THP1, THP2, GLI2/GLI4, GLI3, AW546128, GLI family zinc finger 2, GLI-Kruppel family member GLI2, GLI2, Gli2
    Background
    GLI2 encodes a protein which belongs to the C2H2-type zinc finger protein subclass of the Gli family. Members of this subclass are characterized as transcription factors which bind DNA through zinc finger motifs. These motifs contain conserved H-C links. Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling and they are implicated as potent oncogenes in the embryonal carcinoma cell. The protein encoded by this gene localizes to the cytoplasm and activates patched Drosophila homolog (PTCH) gene expression. It is also thought to play a role during embryogenesis. The encoded protein is associated with several phenotypes- Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial polydactyly type IV, postaxial polydactyly types A1 and B.
    Alias Symbols: HPE9, THP1, THP2
    Protein Interaction Partner: SPOP, SKI, HDAC1, STK36, FBXW11, SUFU, ZIC2, ZIC1, CREB1, TBP,
    Protein Size: 1258
    Molecular Weight
    133 kDa
    Gene ID
    2736
    NCBI Accession
    NM_030379, NP_084655
    UniProt
    P10070
    Pathways
    Hedgehog Signaling, Dopaminergic Neurogenesis
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