Overview

  • Product name
  • Description
    Rabbit polyclonal to L Glutamate
  • Host species
    Rabbit
  • Specificity
    The antibody is calibrated against a spectrum of antigens to assure hapten selectivity. No measurable cross-reactivity (<1:1000) was detected against glutamate in peptides or proteins. Fixed tissue cross-reactivity was tested with known targets at the recommended dilution. No measurable glutaraldehyde-fixed tissue cross-reactivity (<1:1000) was detected against L-alanine, gamma-aminobutyrate, D/L-aspartate, agmatine, guanidine, D/L-arginine, L-citrulline, L-cysteine, D/L-glutamine, glutathione, glycine, L-lysine, L-ornithine, L-serine, taurine, L-threonine, L-tryptophan, L-tyrosine. Modest cross-reactivity (1:20) was detected against D-glutamate. Significant cross-reactivity (1:8) was detected against free NAAG in competition assays (NAAG is not retained in aldehyde-fixed tissue).
  • Tested applications
    Suitable for: IHC-FoFr, IHC-Fr, ICC, Immunomicroscopy, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    L-glutamate conjugated to glutaraldehyde

  • General notes
    The product is optimized for HPI/EHPI with gold or fluorescence detection using etched plastic sections. Filter diluted reagents with 0.2 mm syringe filters before use on EM grids. Enzyme-linked visualizations can be used but will compress the signal dynamic range and are less sensitive.

Properties

Applications

Our Abpromise guarantee covers the use of ab9440 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-FoFr Use at an assay dependent concentration. Use with frozen or vibratome sections is possible but will not yield optimal images as IgGs penetrate aldehyde cross-linked tissue poorly and most amino acids are present at such high levels that prozone effects occur.
IHC-Fr Use at an assay dependent concentration.
ICC 1/100. See protocol. OPTIMAL FIXATION: 0.1-2.5% glutaraldehyde,1% formaldehyde using HPI protocol (The antisera targets the glutaraldehyde conjugate of the hapten). All procedures may be carried out at room temperature. MINIMAL GLUTARALDEHYDE: 0.00% using minimum glutaraldehyde Enhanced HPI (EHPI) protocol with 4% formaldehyde.
Immunomicroscopy Use at an assay dependent concentration.
ICC/IF 1/100.
IHC-P Use at an assay dependent concentration.

Target

  • Relevance
    L Glutamate is a non-essential amino acid naturally occurring in the L-form. It is the most common excitatory neurotransmitter in the Central Nervous System.
  • Alternative names
    • Glutamate antibody
    • L glutamate antibody

Images

  • ab9440 staining L Gluramate in Mouse retina tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with methanol, permeabilized with 0.025% TBS-Triton X-100 and blocked with 5% serum for 1 hour. Samples were incubated with primary antibody (1/100 in PBS + 1% BSA) for 18 hours at 4°C. An Alexa Fluor® 594-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Wu Z  et al. Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzhiemer's disease model. Nat Commun 5:4159 (2014). Read more (PubMed: 24923909) »
  • Li D  et al. Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release. J Physiol 590:855-73 (2012). Read more (PubMed: 22219341) »
See all 7 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Sample
Mouse Cell (retina)
Specification
retina
Permeabilization
Yes - 0.3% TritonX-100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jul 14 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: amb°C
Sample
Mouse Tissue sections (retina)
Specification
retina
Permeabilization
Yes - TBS-Tritòn X-100 0,025 % 15 min
Fixative
Methanol

Dr. Patricia Bogdanov

Verified customer

Submitted Jul 15 2013

Application
Western blot
Sample
Apteronotus leptorhynchus Tissue lysate - whole (Brain)
Loading amount
50 µg
Specification
Brain
Gel Running Conditions
Reduced Denaturing (4-15%)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Mar 05 2013

Question
Answer

Thank you for contacting us.
Here are the references we spoke about on the phone for this product: https://www.abcam.com/L-Glutamate-antibody-ab9440-references.html
Some other background references might be helpful as well to see what other labs have seen for staining.
http://www.ncbi.nlm.nih.gov/pubmed/15215299
http://www.ncbi.nlm.nih.gov/pubmed/18067143
http://cercor.oxfordjournals.org/content/10/11/1132.full
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Apteronotus leptorhynchus Tissue sections (Brain)
Specification
Brain
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
Yes - 0.3% Triton X-100
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jan 24 2013

Answer

Thank you for contacting us and for your interest in our products. I can confirm that ab9440L Glutamate antibody is tested and covered by our guarantee for IHC-FoFr, IHC-Fr, ICC, Immunomicroscopy andIHC-P. I am sorry it has not been tested in ELISA or western blot. All tested applications are listed on the datasheet, and these are updated as soon as any new information is brought to our attention. With regards to publications, the following 2 publications have used the antibody in IHC: Sun D et al. Metabolic and functional profiling of the normal rat retina. J Comp Neurol 505:92-113 (2007). IHC; Rat. PubMed: 17729258 Wilkie IC et al. Physiological and immunocytochemical evidence that glutamatergic neurotransmission is involved in the activation of arm autotomy in the featherstar Antedon mediterranea (Echinodermata: Crinoidea). J Exp Biol 213:2104-15 (2010). IHC-R; Starfish. PubMed: 20511525 I am sorry we have no other L GLutamate antibodies tested in rat that are also tested in WB and ELISA. However, if you would like to test the ab9440 antibody in ELISA or Western blot application, please contact me again prior to the purchase by replying to this message as you may be eligible for our testing discount program. Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates. To find out more about our Abreview system, please see the following link: https://www.abcam.com/abreviews I hope this information is helpful. Please do not hesitate to contact me for any further advice or information.

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Question
Answer

Thanks for your call today and for your patience while I have looked into your question. I found that the antibody ab8893 does not react with free L-glutamate so I don't believe it will be suitable for the ELISA assay that you are planning. We do have a couple of L-glutamate antibodies, ab9440 and ab62668, that do not react with glutamate in peptides or proteins, so it will be specific for the free amino acid. However, we have not tested these antibodies in ELISA. We do have a testing discount program if you would like to try either of them in ELISA, and I would be happy to send more information about this program if you would like. Please let me know if you have any questions, or if there is anything else that we can do for you.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (spinal cord)
Specification
spinal cord
Fixative
Paraformaldehyde
Antigen retrieval step
None
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Mr. Travis Lyttle

Verified customer

Submitted Jan 08 2007

Question

hi Jennifer. Here are my responses to your questions: For ease of reading, i put my answers in CAPS. Just as a reminder, I triple-checked all of our secondary antibodies as well as our glutamate antibody. There is no doubt as to the identity of the secondary antibodies. AB9440-500 LOT#19116 1. Please describe the problem (high background, no staining etc). MY ISSUE IS THAT THERE APPEARS TO BE A SIGNAL WHERE THERE SHOULDN'T BE ONE. THIS IS A RABBIT POLYCLONAL ANTIBODY, BUT IT ONLY SEEMS TO BIND TO OUR GOAT ANTI-MOUSE SECONDARY ANTIBODIES. BACKGROUND IS NOT HIGH AT ALL; COMPARABLE TO "NO PRIMARY ANTIBODY" CONTROLS. 2. On what material are you testing the antibody in IHC? Species? MOUSE •Cell line? R1 ROSA ES CELL-DERIVED NEURONS Tissue? ADULT BRAIN 3. How did you fix the samples? Ethanol, methanol NO Acetone NO Paraformaldehyde YES (4%) Other NO 4. Did you apply antigen retrieval step?NO Enzymatic method NO Heat mediated technique NO Other NO 5. How did you block the unspecific binding sites? 5% NORMAL GOAT SERUM IN .01%TRITON X-100 IN PBS 6. Primary antibody Specification (in which species was it raised against)? RABBIT At what dilution(s) have you tested this antibody? ABCAM'S RECOMMENDED 1:100 Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? INCUBATED OVERNIGHT AT ROOM TEMP IN HUMIDIFIED CHAMBER, FOLLOWED BY 3 PBS RINSES FOLLOWED BY 3 TEN MINUTE PBS WASHES 7. Secondary antibody What secondary antibody are you using? ALEXAFLUOR GOAT ANTI RABBIT IGG 568 OR, FOR EXPERIMENTAL PURPOSES, ALEXAFLUOR GOAT ANTI-MOUSE IGG 488 (WHICH SEEMS TO BE THE ONLY SECONDARY THAT BINDS TO THE PRIMARY AB) Specification (in which species was it raised against)? RABBIT At what dilution(s) have you tested this antibody? 1:100, 1;500; 1:1000 Incubation, wash steps? SAME AS FOR PRIMARY ANTIBODY RINSES AND WASHES Do you know whether the problems you are experiencing come from the secondary? NO. I TESTED OTHER RABBIT POLYCLONAL ANTIBODIES WITH OUR SECONDARIES AND THEY WORKED FINE. What detection method are you using? FLUORESCENCE MICROSCOPY (FIT-C AND TEXAS RED FILTERS) 8. Background staining Please provide an image of your staining 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? ONLY NEGATIVE CONTROL "NO PRIMARY ANTIBODY" 11. Optimization attempts How many times have you tried the IHC? 4 TIMES Do you obtain the same results every time? YES What steps have you altered? ONLYTHE SECONDARY ANTIBODIES. LIKE I SAID, THE GOAT ANTIRABBIT SECONDARY DOESN;T APPEAR TO BIND, WHILE THE GOAT ANTI MOUSE SECONDARY DOES.

Read More
Answer

I have received some further information regarding this antibody from the priginator. Ab9440 does not bind any anti-mouse product that has been tested. The only possible explanation is that the antibody was denatured and that it exposed a non-specific heavy chain motif that the anti-mouse IgG likes, but disrupted the anti-rabbit IgG motif. Any tissue binding was likely non-specific. Some protocol notes that I received - Ab9440 was explicitly designed for glutaraldehyde (GA) fixation and plastic-section detection, especially ultrastructure (e.g. Ottersen 1989 Postembedding Immunogold Labeling of Fixed Glutamate - an Electron-Microscopic Analysis of the Relationship between Gold Particle Density and Antigen Concentration Journal Of Chemical Neuroanatomy 2:57-66). OPTIMAL FIXATION: 0.1-2.5% glutaraldehyde,1% formaldehyde using HPI protocol (please see the protocols tabe on the online datasheet). The residual free glutamate can be detected with FA if the protocol is optimized (>48h fixation, proper temp, pH), yielding enough time for slow methine-bridge formation. See Hayat 1981 Fixation for electron microscopy; Hayat 2001 Principles and Techniques of Electron Microscopy: Biological Applications). If fixation is brief, most or all of the glutamate will be lost. Without GA fixation, over 95% of all amino acids will be lost. Glutamate signal strength will decrease nearly 2 log units. If cells are loaded with 3H-glutamate and fixed with FA, virtually all the free 3H-glutamate is lost. In addition, it is recommend to use a positive tissue control with GA fixation and resin sections - retina, brain, spinal cord will all have strong signals. As I mentioned in my previous email, I can send you a different batch to try. Please let me know if you would like to try it and please send me the Abcam order number or purchase order number that was used when you originally purchased ab9440.

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Question
Answer

Thank you for your enquiry. Unfortunately, we are not aware of any publications that feature the use of this antibody. When we are aware of such references, we cite them on the online datasheets. If you have any more questions, please contact us again.

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1-10 of 12 Abreviews or Q&A

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