Glutamate antibody

Antigen: Glutamate

Clonality: Monoclonal

Host: Mouse

Application: Immunocytochemistry (ICC)

Catalog no.: ABIN99783

Additional Information

Immunogen: Synthetic L-Glutamate conjugated to protein carrier (Pc)

Format: Lyophilized

Isotype: IgM, kappa chain  (Matching secondary antibodies)

Specificity: Conjugated L-Glutamate . Using a conjugate L-Glutamate-(Pc), antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) L-Glutamate-G-(Pc) 1 D-Glutamate-G-(Pc) 1/100 L-Aspartate-G-(Pc) 1/50,000 GABA-G-(Pc) 1/50,000 (a): L-Glutamate-G-(Pc) concentration/other conjugated aminoacids concentration at half displacement G = Glutaraldehyde, GABA = Gamma-Aminobutyric Acid

Application Details

Application Notes: Example of Immunocytochemistry protocol Detection of conjugated L-Glutamate in rat brain 1- Perfusion: The rat is anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the following solutions: Solution A: cacodylate 0.1M, sodium metabisulfite 10g/l, pH = 6.2 Solution B: cacodylate 0.1M, sodium metabisulfite 10g/l and glutaraldehyde 3-5%, pH = 7 2- Post fixation: 15 to 30 min in solution B, then 4 soft washes in Tris 0.05M with sodium metabisulfite 8.5g/l, pH 7.5 (solution C). 3- Tissue sectioning: Cryostat or vibratome sections can be used. 4- Reduction step: Sections are reduced with the solution C containing sodium borohydride (0.1M) for 10 min. Then, the sections are washed 4 times with solution C without sodium borohydride. 5- Application of anti-conjugated L-Glutamate antibody: The final dilution is 1/1,000 to 1/5,000 in solution C containing 0,1% triton X100, plus 2% of non-specific serum. A dozen of sections can be incubated with 2ml of monoclonal antibody solution overnight at 4°C. Then, after this period, the sections are washed 3 times (10 min) with solution C. N.B.: The antibody may be used at a higher dilution. The customer should explore the further antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. 6- PAP procedure: Second antibody: Sections are incubated with 1/200 dilution of goat anti-mouse in solution C for 3 hours at 20°C or 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C PAP: Sections are incubated with 1/1,000 dilution of mouse peroxidase/anti-peroxidase complex in solution C for 1 hour at 37°C. Then, they are washed 3 times (10 min) with solution C Revelation: Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated 0.05% of H 2 O 2 is added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M. Gemacbio sells other anti-catecholamine antibodies raised in rabbit: used together, these tools could be helpful for immunocytochemistry double labelling. Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)

Purity: The ascitic fluid was purified by ammonium sulfate precipitation

Storage: Lyophilized antiboby is stable at least 2 years. After reconstitution with 50 µl of distilled water and 50 µl of glycerol, the aliquot can be repeated freezed (up to five times).

Research Area: Amino Acids

Restrictions: For Research Use only

Publications

Publications: Chagnaud, Campistron, Geffard: "Monoclonal antibody directed against glutaraldehyde conjugated glutamate and immunocytochemical applications in the rat brain." in: Brain research, Vol. 481, Issue 1, pp. 175-80, 1989 (PubMed).

Sinakevitch, Farris, Strausfeld: "Taurine-, aspartate- and glutamate-like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body." in: The Journal of comparative neurology, Vol. 439, Issue 3, pp. 352-67, 2001 (PubMed).

Sinakevitch, Sjoeholm, Hansson et al.: "Global and local modulatory supply to the mushroom bodies of the moth Spodoptera littoralis." in: Arthropod structure & development, Vol. 37, Issue 4, pp. 260-72, 2008 (PubMed).

Claus, Chey, Heinrich et al.: "Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus." in: Journal of virology, 2011 (PubMed).