Anti-DGKZ/DGK-zeta antibody [EPR22040-72]

8 product images

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  Immunoprecipitation - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  DGKZ/DGK-zeta was immunoprecipitated from 0.35 mg of Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate with ab239080 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab239080 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/1000 dilution.
  Lane 1: Jurkat whole cell lysat 10 μg (input).
  Lane 2: ab239080 IP in Jurkat whole cell lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab239080 in Jurkat whole cell lysate (-).
  

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  Exposure time: 15 seconds.

  All lanes: Immunoprecipitation - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Predicted band size: 124 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Immunohistochemical analysis of paraffin-embedded rat brain tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on neurons of rat brain (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Immunohistochemical analysis of paraffin-embedded mouse brain tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and nuclear staining on neurons of mouse brain (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and nuclear staining on Purkinje cells of human cerebellum (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on subset of immune cells in human tonsil (PMID: 24573202) is observed. Counter stained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Western blot - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
  

  Exposure time:
  Lane 1-2: 15 seconds.
  Lane 3-8: 3 seconds.

  The lysates were not boiled to prevent the target protein from aggregation. Multiple bands are likely due to isoforms and phosphorylation. The molecular mass of human DGKZ / DGK-zeta is also predicated to be larger than that of rodents. The molecular profile/weight observed is consistent with what has been described in the literature (PMID: 12070163, PMID: 14511325, PMID: 9657393).

  All lanes: Western blot - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080) at 1/1000 dilution

  Lane 1: Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

  Lane 2: HuT-78 (human Sezary syndrome cutaneous T lymphocyte), whole cell lysate at 20 µg

  Lane 3: Human cerebellum tissue lysate at 20 µg

  Lane 4: Mouse brain tissue lysate at 20 µg

  Lane 5: Mouse cerebellum tissue lysate at 20 µg

  Lane 6: Mouse thymus tissue lysate at 20 µg

  Lane 7: Rat brain tissue lysate at 20 µg

  Lane 8: Rat cerebellum tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

  Predicted band size: 124 kDa

  Observed band size: 90-130 kDa

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  Flow Cytometry (Intracellular) - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilzedJurkat (human T cell leukemia cell line from peripheral blood) cell line labeling DGKZ/DGK-zeta with ab239080 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730, Black) isotype control, and an unlabeled control (Cell without incubation with primary antibody and secondary antibody, Blue).
  

  Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 wasused as the secondary antibody.

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  Multiplex immunohistochemistry - Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080)

  Fluorescence multiplex immunohistochemical analysis of mouse cerebellum (formalin-fixed paraffin-embedded section).

  
  Panel A: merged staining of anti-GABRA6 (magenta; Opal™690), anti-KCNA (green; Opal™520) and anti-DGKZ (gray; Opal™570) on mouse cerebellum.
  
  Panel B: anti-GABRA6 staining granule cells in mouse cerebellum.
  
  Panel C: anti-KCNA staining basket cells in mouse cerebellum.
  
  Panel D: anti-DGKZ staining Purkinje cells in mouse cerebellum.
  
  Nuclear DNA was labeled with DAPI (shown in blue).
  

  The section was incubated in three rounds of staining: in the order of Anti-GABA A Receptor alpha 6 antibody [EPR25322-129] ab300069 at a 1/500 dilution, Anti-KCNA antibody [EPR26383-85] ab313624 at a 1/5000 and ab239080 at a 1/5000 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.