Overview

  • Product name

    Anti-GAD65 + GAD67 antibody [EPR19366]
    See all GAD65 + GAD67 primary antibodies
  • Description

    Rabbit monoclonal [EPR19366] to GAD65 + GAD67
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IP, IHC-P, IHC-Fr, WBmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Mouse GAD65 + GAD67 aa 450 to the C-terminus. The exact sequence is proprietary.
    Database link: P48318

  • Positive control

    • WB: Mouse GAD67 and GAD65 fragment recombinant proteins; Human, mouse and rat cerebellum lysates; C6 whole cell lysate; Mouse and rat brain lysates. IHC-P: Mouse cerebellum and pancreas tissues; Rat cerebellum and pancreas tissues. IHC-Fr: Mouse hippocampus tissue.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab183999 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
IP 1/40.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IHC-Fr 1/2500.
WB 1/1000. Detects a band of approximately 65, 67 kDa (predicted molecular weight: 65, 67 kDa).

Target

  • Relevance

    This gene encodes one of several forms of glutamic acid decarboxylase, identified as a major autoantigen in insulin-dependent diabetes. The enzyme encoded is responsible for catalyzing the production of gamma aminobutyric acid from L glutamic acid. A pathogenic role for this enzyme has been identified in the human pancreas since it has been identified as an autoantibody and an autoreactive T cell target in insulin dependent diabetes. This gene may also play a role in the stiff man syndrome.
  • Cellular localization

    Cytoplasm; cytosol. Associated to cytoplasmic vesicles. In neurons, cytosolic leaflet of Golgi membranes and presynaptic clusters.
  • Database links

  • Alternative names

    • 65 kDa glutamic acid decarboxylase antibody
    • 67 kDa glutamic acid decarboxylase antibody
    • GAD 65 antibody
    • GAD 67 antibody
    • GAD1 antibody
    • GAD2 antibody
    • Glutamate decarboxylase 1 antibody
    • Glutamate decarboxylase 2 antibody
    • Glutamate decarboxylase 2 pancreatic islets and brain 65kDa antibody
    • Glutamate decarboxylase 65 kDa isoform antibody
    • Glutamate decarboxylase 67 kDa isoform antibody
    • SCP antibody
    see all

Images

  • All lanes : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

    Lane 1 : Mouse GAD67 fragment recombinant protein
    Lane 2 : Mouse GAD65 fragment recombinant protein

    Lysates/proteins at 0.01 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 65, 67 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1: 1 second; Lane 2: 15 seconds.

    Mouse GAD67 fragment recombinant protein contain aa468-592 with a His-Tag®. Mouse GAD65 fragment recombinant protein contain aa460-584 with a His-Tag®. These two fragment recombinant proteins were made in-house. The ~30 kDa band represents doublets of the recombinant fragments.

  • Immunohistochemical analysis of paraffin-embedded Mouse cerebellum tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse cerebellum is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

  • All lanes : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

    Lane 1 : Human cerebellum lysate
    Lane 2 : Mouse skin lysate
    Lane 3 : Mouse lung lysate
    Lane 4 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
    Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
    Lane 6 : Mouse cerebellum lysate
    Lane 7 : Rat cerebellum lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 65, 67 kDa
    Observed band size: 65,67 kDa
    why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1-5: 3 minutes; Lane 6 and 7: 2 seconds.

  • Ab183999 Immunoprecipitating GAD65 + GAD67 in Human cerebellum lysate. 10µg of cell lysate was incubated with primary antibody 1/40. For western blotting Ab183999 (1/1000) was used to confirm successful immunoprecipation. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000.

    Lane 1 (Input): Human cerebellum lysate 10μg
    Lane 2 (+): Human cerebellum lysate with Ab183999, 1/40
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab183999 in Human cerebellum lysate

  • Lane 1 : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/5000 dilution
    Lane 2 : Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999) at 1/1000 dilution

    Lane 1 : Mouse brain lysate
    Lane 2 : Rat brain lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 65, 67 kDa
    Observed band size: 65,67 kDa why is the actual band size different from the predicted?


    Exposure time: 2 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse pancreas islets is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

  • Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative on mouse lung. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

  • Immunohistochemical analysis of 4% paraformaldehyde fixed, 0.2% Triton X-100 permealized frozen section of Mouse hippocampus tissue labeling GAD65 + GAD67 with ab183999 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) secondary (ab150077) at 1/1000 dilution (green). The result showed mainly cytoplasmic staining on mouse hippocampus. The nuclear counterstain is DAPI (blue).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

  • Immunohistochemical analysis of paraffin-embedded Rat cerebellum tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat cerebellum is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

  • Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat pancreas islet is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

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

  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling GAD65 + GAD67 with ab183999 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative on rat kidney. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

     

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

References

This product has been referenced in:

See 1 Publication for this product

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