Antibodies, Proteins, Kits and Reagents for Life Science

257 980 ₸ Product size

100 µl 257 980 ₸

Order now and get it on Tuesday March 09, 2021

Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR19366] to GAD65 + GAD67
Suitable for: IP, IHC-P, IHC-Fr, WB, ICC/IF
Reacts with: Mouse, Rat, Human, Recombinant fragment

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 & Species

Application	Species
ICC/IF	Mouse
IHC-Fr	Mouse
IHC-P	Mouse Rat
IP	Human
WB	Mouse Rat Human Recombinant fragment

See all applications and species data

Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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. ICC/IF: Mouse primary neuron cells
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR19366
Isotype

IgG
Research areas

Immunocytochemistry/ Immunofluorescence - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

Immunocytochemistry/ Immunofluorescence analysis of mouse primary neuron cells labeling GAD65 + GAD67 with purified ab183999 at 1/100 (10 µg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with ab11267 Anti-MAP2 antibody [HM-2]; ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.
Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunoprecipitation - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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
Western blot - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Frozen sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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.
Anti-GAD65 + GAD67 antibody [EPR19366] (ab183999)

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