Dihydroxyphenylalanine, L- anticorps

Détails pour le produit réf. ABIN6559947
Antigène
Reactivité
Toutes les espèces
1
Hôte
Lapin
1
Clonalité
Polyclonal
Application
Immunocytochemistry (ICC)
1
1
1
Options
Immunogène Synthetic L-DOPA conjugated to protein carrier (Pc)
Isotype IgG
Specificité Conjugated L-dihydroxyphenylalanine (L-DOPA) . Using a conjugate L-DOPA-(Pc), antibody specificity was performed with an ELISA test by competition experiments with the following compounds: Compounds Cross-reactivity ratio (a) L-DOPA-G-(Pc) 1 -methyl-L-DOPA-G-(Pc) 1/2,200 3-O-methyl-L-DOPA-G-(Pc) 1/>50,000 Dopamine-G-(Pc) 1/>50,000 Noradrenaline-G-(Pc) 1/>50,000 Tyrosine-G-(Pc) 1/>50,000 (a): L-DOPA-G-(Pc) concentration /other conjugated catecholamine concentration at half displacement G = Glutaraldehyde
Purification Antiserum previously preabsorbed on protein carriers, and purified
Antigène
Classe de substances Amino Acid
Indications d'application Example of cytochemistry application Detection of conjugated L-DOPA in rat brain 1- Perfusion: The rat is anaesthetized 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 = 7.5 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 sectionning: 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-DOPA antibodies: The final dilution is 1/1,000 to 1/5,000 in solution C containing triton X100 0.5%, plus 2% of non-specific serum. A dozen of sections can be incubated with 2ml of antibody solution overnight at 4°C. Then, after this period, the sections are washed 3 times (10 min) with solution C. N.B.: Antibodies may be used at a higher dilution. The customer should explore the 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 1/100 dilution of goat anti-rabbit 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 rabbit 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 the same and other antibodies to conjugated small molecules raised in mouse:used together, these tools could be helpful for immunocytochemistry double labelling. Double detection of conjugated L-DOPA and Dopamine in rat brain 1- Perfusion: The rat will be deeply anesthetized with sodium Pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with the 500ml of 5% glutaraldehyde (G), 0.9% sodium metabisulfite (SMB) solution in 0.1cacodylate buffer pH 7,4. 2- Post fixation: 2h, 4°C in the same fixative solution. 3-Tissue sectionning: Cryostat or vibratome sections can be used. 4- Application of anti-conjugated antiserum: Sections will be reduced in 0.05M Tris buffer containing 0.9% SMB (Tris- SMB). Then, the sections will be washed in the same solution (12h, 4°C) and incubated in Tris-SMB containing 3% non specific serum and 0.1% Triton X100 (8h at 4°C). 5- Application of anti-conjugated L-DOPA antibodies: Free floating adjacent sections will be incubated (24h, 4°C) with a polyclonal antiserum against conjugated L-DOPA (1/,1000 to 1/5,000), with a monoclonal antibody against conjugated DA (1/,1000 to 1/5,000), and with both. Antisera will be diluted in Tris-SMB, 1% non-specific serum, 0.2% Triton X100 solution. N.B.: Antibodies may be used at a higher dilution. The customer should explore the 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. Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)
Restrictions For Research Use only
Format Lyophilized
Stock 4 °C