Anti-GAD65 antibody [GAD-6] (ab26113)

Reviews (1)Q&A (7)References (32)

Overview

  • Product name
    Anti-GAD65 antibody [GAD-6]
    See all GAD65 primary antibodies
  • Description
    Mouse monoclonal [GAD-6] to GAD65
  • Host species
    Mouse
  • Specificity
    The GAD-6 monoclonal antibody ab26113 recognizes only GAD65 and has no cross reactivity with GAD67 on western blots of mouse brain or purified recombinant GAD67 and GAD65. ab26113 recognizes GAD65 primarily in nerve terminals in immunohistochemical analysis of brain sections (Kaufman et al, 1991).
  • Tested applications
    Suitable for: IHC-P, WB, IHC-Fr, IHC-FoFr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Mammals
  • Immunogen

    Full length protein (Rat)

  • Epitope
    ab26113 recognizes an epitope near the C-terminus of GAD65
  • Positive control
    • mouse brain

Properties

Applications

Our Abpromise guarantee covers the use of ab26113 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
IHC-P Use at an assay dependent concentration. PubMed: 18467596
WB 1/1000 - 1/20000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
IHC-Fr Use at an assay dependent concentration.
IHC-FoFr 1/100. PubMed: 24246263
ICC/IF Use at an assay dependent concentration. PubMed: 22745816

Target

  • Function
    Catalyzes the production of GABA.
  • Sequence similarities
    Belongs to the group II decarboxylase family.
  • Post-translational
    modifications
    Phosphorylated; which does not affect kinetic parameters or subcellular location.
    Palmitoylated; which is required for presynaptic clustering.
  • Cellular localization
    Cytoplasm > cytosol. Cytoplasmic vesicle. Cell junction > synapse > presynaptic cell membrane. Golgi apparatus membrane. Associated to cytoplasmic vesicles. In neurons, cytosolic leaflet of Golgi membranes and presynaptic clusters.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • 65 kDa glutamic acid decarboxylase antibody
    • DCE 2 antibody
    • DCE2 antibody
    • DCE2_HUMAN antibody
    • GAD 2 antibody
    • GAD 65 antibody
    • GAD-2 antibody
    • GAD-65 antibody
    • Gad2 antibody
    • Glutamate decarboxylase 2 (pancreas) antibody
    • Glutamate Decarboxylase 2 (pancreatic islets and brain 65kDa) antibody
    • Glutamate decarboxylase 2 antibody
    • Glutamate Decarboxylase 65 antibody
    • Glutamate decarboxylase 65 kDa isoform antibody
    • Glutamic Acid Decarboxylase 2 antibody
    • Glutamic Acid Decarboxylase 65 antibody
    • MGC161605 antibody
    • MGC161607 antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 antibody [GAD-6] (ab26113)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAD65 antibody [GAD-6] (ab26113)
    Mouse monoclonal [GAD-6] to GAD65 staining GAD65-containing nerve terminals around cell bodies of Purkinje cells in the rat cerebellar cortex.
  • Western blot - Anti-GAD65 antibody [GAD-6] (ab26113)
    Western blot - Anti-GAD65 antibody [GAD-6] (ab26113)
    Anti-GAD65 antibody [GAD-6] (ab26113) at 1/1000 dilution + mouse cerebellum at 0.75 µg

    Secondary
    HRP horse anti-mouse IgG at 1/5000 dilution

    Predicted band size: 65 kDa

  • Immunohistochemistry (Frozen sections) - Anti-GAD65 antibody [GAD-6] (ab26113)
    Immunohistochemistry (Frozen sections) - Anti-GAD65 antibody [GAD-6] (ab26113)Winpenny et al, Neural Development, 6, 12, Fig. 4f.; doi: 10.1186/1749-8104-6-12

    Immunohistochemical analysis of frozen section from Mouse embryo, staining the glomerular layer of the brain. GAD65 is labelled with ab26113 (green) diluted 1:1000. Trb2 is labelled with ab23345 (red).

  • Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-GAD65 antibody [GAD-6] (ab26113)
    Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-GAD65 antibody [GAD-6] (ab26113)This image is courtesy of an Abreview submitted by Patrick Ye.

    Immunohistochemical analysis of Mouse brain cortex sections, labelling GAD65 with ab26113, diluted 1/100.

    See Abreview

References

This product has been referenced in:
  • Woodhouse A  et al. Repeat propofol anesthesia does not exacerbate plaque deposition or synapse loss in APP/PS1 Alzheimer's disease mice. BMC Anesthesiol 18:47 (2018). Read more (PubMed: 29699479) »
  • Hwang S  et al. Hypoxia regulates the level of glutamic acid decarboxylase enzymes and interrupts inhibitory synapse stability in primary cultured neurons. Neurotoxicology 65:221-230 (2018). WB . Read more (PubMed: 29107683) »
See all 32 Publications for this product

Publishing research using ab26113? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Western blot abreview for Anti-GAD65 antibody [GAD-6]

 Excellent
Abreviews
Application
Western blot
Sample
Human Tissue lysate - whole (human hippocampus)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
10 µg
Treatment
used protease inhibitors
Specification
human hippocampus
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
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Abcam user community

Verified customer

Submitted Jun 25 2015

Question



Inquiry: Hello, Do you have an antigen for pre-adsorption controls for teh following product? https://www.abcam.com/GAD65-antibody-GAD-6-Neuronal-Marker-ab26113.html

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Answer

Thank you for the clarification! Unfortunately the blocking peptide is not available for this particular antibody. Please let me know if I can be of further assistance.

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Question


Thank you for replacement GAD65 ab 112497. I try it on my western blot. The ab
lind up to all my mark. and no band was found on my positive control(Rat brain
tissue). The reason I select 112497, because I think this is polycolonal ab and
may give mpre chance for my cells.
If you can send me another GAD 65 ab 75750, I can try!
Thanks!

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Answer

Thank you very much for your reply.

I am sorry to hear that the replacement ab112497 did not work in Western blot. Just to confirm, there were multiple bands in your neuron lysate, and no bands with the rab brain lysate? Were you using the same protocol as before?

I'm sending a replacement vial of ab75750 on the order ***, which we expect to arrive around Thursday of next week. Please keep me updated about the results with this antibody as well. If you have any questions or need anything else, please let me know and I'll be happy to help.

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Question


Thank you for your information.
I didn't try it in other Rat tissues, I may try it and let you know the results.
I also couldn't find any related papers.
I would like to try polycolone Ab.
Best Regards!

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Answer

Thank you very much for your reply.

I'm sending a free of charge vial of ab112497 on the order **** so that you can compare the results to those with ab26113. This antibody is out of stock but we expect it to arrive around September 27 (next Thursday). For our records, could you also let me know if you block with milk or BSA during your Western blot experiments?

Please keep me updated about the results with ab112497 once you've had a chance to try it. I look forward to hearing from you. If there is anything else that we can do for you please let me know and I'll be happy to help.

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Question

Band around 65 kDa with rat brain tissue lysate, 40 kDa in neural cell lysate. Is this 40 kDa band specific. 1:1000 dilution. Normal culture conditions.

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Answer

Thank you very much for your call yesterday and for your patience while I've looked into your enquiry.

I actually could not find a reference showing analysis of endogneous GAD65 in culture neurons by Western blot. So, I'm still not quite sure how to explain this 40 kDa band in your samples. Do you have access to any other tissues to use as a positive control, just to further verify that the antibody is binding specifically? Have you managed to find any information in the literature?

I am very sorry that I don't have more information, but if you have any concerns about this antibody and feel that it is binding non-specifically, I'd be happy to send you a replacement antibody such as ab75750 or ab112497-

https://www.abcam.com/GAD65-antibody-EPR2553Y-ab75750.html

https://www.abcam.com/GAD65-antibody-ab112497.html

I look forward to hearing from you. Please let me know if you have any questions or need anything else from me.

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Question

Hello,



thanks a lot for your answer.

I would have another question though: as the GAD65 antibody is confined to nerve terminals, is it (in general) possible to stain for GAD65 cell bodies? Are there antibodies that are specifically used to stain for these cell bodies? I mean in the literature you see stainings for GAD65 cell bodies..



My best,

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Answer

Thank you for contacting us.

We are happy to help customers find the most suitable products for their research purposes; however we are not specialized in this particular field and do not want to recommend a product that may be unsuitable. We encourage customers to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about their specific research interests.

I am sorry that I could not be more helpful, but I hope that the available literature in this area can provide some clarification. Please do not hesitate to contact us again with other needs or with any questions about our products.

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Question

Lot number: Gr56581-1
Inquiry: Hello, in our lab we want to stain for GAD65 neuronal cell bodies in mouse spinal cord (see protocol attached). As described in the literature and our protocol, we leave out any Triton steps for this. The staining works (labeling found mainly in Rexed lamina 1 and 2) but as besides the GAD65 cell bodies a lot of synapses around are labeled as well, we hardly see them! Do you know a method or a special treatment to prevent synaptic labeling in this case? For GAD67 this kind of staining works nicely (cell bodies clearly visible). Is there maybe a difference in the staining because GAD65 is supposed to be located more in synapses (in comparison to GAD67)? But as in the literature GAD65 cell body staining have been described, it should somehow be possible to make them visible in a clear way. Thanks in advance,

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Answer

Thank you for your enquiry regarding ab26113.

It has been reported that GAD65 is transported to nerve terminals and this antibody (ab26113) should stain them as “puncta” around the cell body of their target neurons.

On the other hand, GAD67 (ab26116) is localized to cell bodies, axons and terminals and it will stain these well as long as the samples undergo antigen retrieval.

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question

DESCRIPTION OF THE PROBLEM No signal SAMPLE Neurodifferentiated human embryonic stem cells PRIMARY ANTIBODY Abcam (ab26113), Mouse monoclonal to GAD65, in Knouckout Serum replacement @ 1:500, incubate for 1 hour and wash 3 times with 100 uL PBS ++ DETECTION METHOD GE Healthcare In Cell 6000, High content analyzer using confocal imaging POSITIVE AND NEGATIVE CONTROLS USED As this is a routine procedure, we do not use positive and negative controls on every plate. This assay plate was stained for other markers which worked fine indicating that there was no problem with the staining method or the image capture system. We have previously used this antibody in this manner and have gotten good results. We expect our cultures to be 60-70% GAD65 positive. We have in the past run a negative control (undifferentiated stem cells). Previous run (GAD65) in red ANTIBODY STORAGE CONDITIONS stored at -20C in 10 uL aliquots FIXATION OF SAMPLE 10% formalin (Sigma-Aldrich ANTIGEN RETRIEVAL None PERMEABILIZATION STEP Yes, 0.25% triton x-100 BLOCKING CONDITIONS No blocking SECONDARY ANTIBODY Invitrogen (A31571), Donkey anti-mouse IgG Alexa Fluor 647 , in Knouckout Serum replacement @ 1:500, incubate for 1 hour and wash 3 times with 100 uL PBS ++ HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for contacting Abcam.

Based on the protocol the customer sent, there are a few suggestions that may help resolve the issue:

1 - Increase the concentration of primary antibody used to 1/200.

2 - Increase the incubation time to 3 hours at room temperature.

I understand that the customer had previously purchased vials of this antibody and it has proved to be successful for them but as we have not tested ab26113 in either human cells or if it is not covered under our Abpromise. Please let me know if changing the above does not prove to be successful and I will if there is anything else I can do.

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