Overview

  • Product name
    Anti-GAD67 antibody [K-87]
    See all GAD67 primary antibodies
  • Description
    Mouse monoclonal [K-87] to GAD67
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, IHC-P, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide:

    RFRRTETDFSNLFARDLLPA

    , corresponding to amino acids 87-106 of Human GAD67

  • Positive control
    • Recombinant Human GAD67 protein (ab114255) can be used as a positive control in WB. In WB, this antibody gave a positive signal in Mouse and Rat Brain Tissue Lysates. GAD67 has been thought to be primarily located in the nerve cell body, but using this new K-87 mAb, GAD67 can now also be detected in dendrites and axons.
  • General notes


    ab26116 mouse monoclonal [K-87] to GAD67 specically recognizes GAD67 and has no cross reactivity with GAD65; this has been shown on western blots of mouse brain or purified recombinant GAD67 and GAD65. In immunohistochemical analysis of brain sections, the K-87 monoclonal recognizes GAD67 in nerve cell bodies and has an enhanced ability to detect GAD67 in dendrites and axon buttons compared to the original anti-GAD67 K-2 polyclonal antibody (see Kaufman et al, 1991).

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.01% Sodium azide
    Constituent: 0.0268% PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Primary antibody notes
    ab26116 mouse monoclonal [K-87] to GAD67 specically recognizes GAD67 and has no cross reactivity with GAD65; this has been shown on western blots of mouse brain or purified recombinant GAD67 and GAD65. In immunohistochemical analysis of brain sections, the K-87 monoclonal recognizes GAD67 in nerve cell bodies and has an enhanced ability to detect GAD67 in dendrites and axon buttons compared to the original anti-GAD67 K-2 polyclonal antibody (see Kaufman et al, 1991).
  • Clonality
    Monoclonal
  • Clone number
    K-87
  • Isotype
    IgG1
  • Light chain type
    kappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab26116 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
ICC/IF Use a concentration of 10 µg/ml.
Flow Cyt Use 1µg for 106 cells.

(methanol fixed cells)
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-P 1/1000 - 1/10000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. An antigen retrieval step is necessary (e.g. incubating the sections at 90 degrees C in a 50mM citrate buffer). Cell body labeling is optimized when Triton is omitted from the tissue processing. Axon terminal labeling is substantially increased (and cell body labeling decreased) when Triton is included (see Soghomonian et al, 1998).
WB 1/1000 - 1/20000. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).
IHC-Fr 1/2000.

Target

  • Function
    Catalyzes the production of GABA.
  • Tissue specificity
    Isoform 3 is expressed in pancreatic islets, testis, adrenal cortex, and perhaps other endocrine tissues, but not in brain.
  • Involvement in disease
    Defects in GAD1 are the cause of cerebral palsy spastic quadriplegic type 1 (CPSQ1) [MIM:603513]. A non-progressive disorder of movement and/or posture resulting from defects in the developing central nervous system. Affected individuals manifest symmetrical, non-progressive spasticity and no adverse perinatal history or obvious underlying alternative diagnosis. Developmental delay, mental retardation and sometimes epilepsy can be part of the clinical picture.
  • Sequence similarities
    Belongs to the group II decarboxylase family.
  • Information by UniProt
  • Database links
  • Alternative names
    • 67 kDa glutamic acid decarboxylase antibody
    • CPSQ1 antibody
    • DCE1 antibody
    • DCE1_HUMAN antibody
    • EC 4.1.1.15 antibody
    • FLJ45882 antibody
    • GAD 67 antibody
    • GAD antibody
    • GAD-67 antibody
    • GAD1 antibody
    • Glutamate decarboxylase 1 (brain, 67kDa) antibody
    • Glutamate decarboxylase 1 antibody
    • Glutamate decarboxylase 1 brain 67kD antibody
    • Glutamate decarboxylase 1 brain 67kDa antibody
    • Glutamate decarboxylase 67 kDa isoform antibody
    • Glutamate decarboxylase, brain, 67-KD antibody
    • OTTHUMP00000041055 antibody
    • SCP antibody
    see all

Images

  • Anti-GAD67 antibody [K-87] (ab26116) at 1/2000 dilution + mouse cerebellum at 0.75 µg

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

    Predicted band size: 67 kDa

  • ab26116, at a 1/2000 dilution, staining of fixed mouse cerebellum sections that underwent an antigen retrieval step.
  • Immunohistochemical staining of mouse pancreatic islet beta cells after antigen retrieval with ab26116 at a dilution of 1/1000.

  • ICC/IF image of ab26116 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab26116, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Overlay histogram showing PC12 cells stained with ab26116 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab26116, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in PC12 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.

    Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

References

This product has been referenced in:
  • Tan W  et al. Neuronal loss in anterior cingulate cortex in spared nerve injury model of neuropathic pain. Neurochem Int 118:127-133 (2018). WB . Read more (PubMed: 29894700) »
  • Mukherjee A & Caroni P Infralimbic cortex is required for learning alternatives to prelimbic promoted associations through reciprocal connectivity. Nat Commun 9:2727 (2018). Read more (PubMed: 30006525) »
See all 41 Publications for this product

Customer reviews and Q&As

1-10 of 27 Abreviews or Q&A

Question

Dear sir/madam;
Thanks for your kind reply and keen support.
These are my answers regarding the questionnaire you asked me to fill about the problem with my antibody;

Order Details
Antibody code:
Anti-GAD67 antibody [K-87] - Neuronal Marker (ab26116)
Lot number:
GR38591-1
Purchase order number
or preferably Abcam order number:
Purchase Order Number: #####


General Information
Antibody storage conditions (temperature/reconstitution etc)
Aliquots (10 µl each) were stored in -20 freezer.
Description of the problem (high background, low signal, non-specific staining etc.)
Non-specific staining of almost all cells.

Sample (Species/Tissue/Cell Type/Cell Line etc.)
1. Paraffin sections of human foetal brain.
2. Frozen sections of human foetal brain.
3. Frozen sections of mouse brain.

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Paraffin sections were processed according to the standard tissue processing protocols. Fixation was done with 4% Para formaldehyde in PBS @ 4°C then in 70% Ethanol.
Frozen sections were either non-fixed or pre-fixed with 4% Para formaldehyde.

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Heat induced antigen retrieval (microwave) in 10mM Na citrate buffer, pH 6.0

Permeabilization step
0.1% Triton-x-100 / PBS (PBST) solution was used for permeabilization.

Blocking conditions (Buffer/time period, Blocking agent etc.)
Blocking was done in 5% normal horse serum in 0.1% Triton-x-100 / PBS for 15-30 minutes at room temperature.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Abcam, Mouse monoclonal Anti-GAD67 antibody [K-87] - Neuronal Marker, Dilution started with either PBS or PBST in 1:1000 then decreasing gradually into 1:2000, 1:5000, and 1:10000, incubation was done overnight @ 4°C, washes were done 3 times for 5 minutes each with PBST or PBS.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Vector laboratories, Biotinylated Horse Anti-Mouse IgG Antibody, dilution was 1:200 (in PBST), incubation was done either for 2 hours @ 4°C or 1 hour at room temperature.

Detection method
ImmpactTM Chromogen.

Positive and negative controls used (please specify)
Positive controls were done with TBR1 antibody (from Abcam).
Negative controls were done with no primary antibody.

Optimization attempts (problem solving)
Increasing the dilution, Changing the time of heating step, increasing blocking step duration, increasing washes duration, and decreasing the secondary antibody incubation.

How many times have you tried the IHC?
6 times in a total of 24 slides.

Have you run a No Primary control?
Yes
Do you obtain the same results every time?
Yes
What steps have you altered?
Increasing the dilution, changing the time of heating step, increasing blocking step duration, increasing washes duration, and decreasing the secondary antibody incubation.
We would appreciate if you are also able to provide and image which would help us to assess the results
See attached files.

Read More
Answer

Thank you for taking the time to complete our questionnaire.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab26116. I would also appreciate if you can confirm some further details:

1. Could you confirm how long was the antigen retrieval? I can recommend totry different time points if this hasn't already been tried, as this can sometimes require optimization. For example, try 2, 5, 10, 15 and 20 minutes.

2. Could you confirm how long was permeabilisation in Triton? Triton is quite a strong detergent, so we would recommend 10 minutes only.

3. I can recommend to use a more gentle detergent such as 0.2% tween in the other buffers rather than Triton. Also to include 0.2% Tween in the wash bufferwhich will help to wash away any excess antibody.

4. Could you confirm if the secondary working well with other mouse IgG1 primary antibodies? What were the results of the no primary control?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested informationand details of how you would like to proceed.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Dog Cell (neuron)
Permeabilization
Yes - TritonX 0.05%
Specification
neuron
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 15 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (brain tissue)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate ph 6
Permeabilization
No
Specification
brain tissue
Blocking step
Normal Goat Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 26°C
Fixative
Formaldehyde

Herr Dr. Markus Kipp

Verified customer

Submitted Sep 07 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Guinea pig Tissue sections (Brain tissue ( Hippo section))
Antigen retrieval step
Other
Permeabilization
No
Specification
Brain tissue ( Hippo section)
Blocking step
BSA+Goat serum+triton as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.4% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 06 2018

Application
Western blot
Sample
Rat Tissue lysate - whole (whole brain)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
10 µg
Specification
whole brain
Blocking step
BSA as blocking agent for 4 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Brian Pham

Verified customer

Submitted Jan 05 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Brain, cortex)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate
Permeabilization
No
Specification
Brain, cortex
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 1%
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 07 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (brain)
Antigen retrieval step
None
Permeabilization
Yes - PBSTx 3x5mn rt
Specification
brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
Fixative
Paraformaldehyde

Yacine Tensaouti

Verified customer

Submitted Aug 02 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Cell (Brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 20mM Citrate
Permeabilization
Yes - Tween20 0.2%
Specification
Brain
Blocking step
normal horse serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Apr 19 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Astatotilapia burtoni Tissue sections (Brain)
Permeabilization
Yes - 0.1% Triton
Specification
Brain
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 02 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (mouse brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 50mM citrate buffer
Permeabilization
No
Specification
mouse brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted May 06 2015

1-10 of 27 Abreviews or Q&A

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