The transmembrane dopamine transporter (DAT) is located on the presynaptic nerve terminal and is responsible for terminating dopaminergic transmission by transporting dopamine from the synaptic cleft into the dopaminergic neuron (reuptake). Dopaminergic pathways have been strongly implicated in reward and addiction, motivation, alcoholism, ADHD and degenerative motor diseases such as Parkinson′s, Huntington′s and Chorea.
Recognizes Dopamine transporter, N-terminus. Shows no cross reactivity to the closely related serotonin and norepinephrine transporters (Miller, 1997). Immunolocalization of DAT on paraformaldehyde fixed frozen sections of human brain using MAB369 shows dense punctate staining throughout the caudate, putamen and accumbens (Miller, 1997).
N-terminus of human dopamine transporter fused to Glutathione S-transferase.
Western blot: Recognizes a diffuse band at approximately 70-85 kDa on western blots of extracts (20 ug total protein) from human caudate, putamen, and nucleus accumbens.
Immunohistochemistry: 4% paraformaldehyde fixed tissue (care should be taken not to over-fix tissue); perfusion followed by less than 90 minutes post-fixation, then cryoprotect. Suggested working dilution 1:1,000-1:10,000. If using on rat tissue, absorbed anti-rat secondary antibodies are recommended, and the use of rat PAP systems, or ABC detection will enhance sensitivity.
Immunocytochemistry: 1:5,000 to 1:10,000.
Immunoblotting (not recommended for use on rat)
Optimal working dilutions and protocols must be determined by end user.
IMMUNOHISTOCHEMISTRY PROTOCOL FOR MAB369
This antibody has been used successfully on 30 μm, free floating, 4% paraformaldehyde fixed mouse brain tissue. All steps are performed under constant agitation. Suggested protocol follows.
1) 3 x 10 minute washes in TBS (with or without 0.25% Triton).
2) Incubate for 30 minutes in TBS with 3% serum (same as host from secondary antibody).
3) Incubate primary antibody diluted appropriately in TBS with 1% serum (same as host from secondary antibody) (with or without 0.25% Triton) for 2 hours at room temperature followed by 16 hours at 4°C.
4) 3 x 10 minute washes in TBS.
5) Incubate with secondary antibody diluted appropriately in TBS with 1% serum (same as host from secondary antibody).
6) 3 x 10 minute washes in TBS.
7) ABC Elite (1:200 Vector Labs) in TBS.
8) 2 x 10 minute washes in TBS.
9) 1 x 10 minute wash in phosphate buffer (no saline).
10) DAB reaction with 0.06% NiCl added for intensification.
11) 2 x 10 minute washes in PBS.
12) 1 x 10 minute wash in phosphate buffer (no saline).
This Anti-Dopamine Transporter Antibody, N-terminus, clone DAT-Nt is validated for use in IC, IH, WB for the detection of Dopamine Transporter.
Research Sub Category
Ion Channels & Transporters
~ 70-85 kDa
UnPurified tissue culture supernatant, filtered through a 0.2 μm membrane prior to vialing. Product contains 20%FBS and Ciprofloxacin at final concentration of 10μg/mL.
Storage and Stability
Maintain for 1 year at -20°C from date of shipment. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Positive Control: Brain (caudate, putamen, and nucleus accumbens) tissue
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
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