Immunofluorescence was performed on sections of adult rat pineal. Tissue sections on slides were probed for 24 h at 4°C in a humidified chamber with rabbit anti-SNAT (Product # PA1-762) at an antibody concentration of 5-10 µg/ml diluted in 50 mM Tris-HCl, pH 7.4, containing 1.5% NaCl, 0.3% Triton X-100 (TBST) and 4% normal goat serum. After overnight incubation, sections were washed extensively for 1 h in TBST, and detection of primary antibody was performed for 1.5 h at room temperature with AlexaFluor-488-conjugated donkey anti-rabbit diluted 1:600 in TBST. Sections were then washed in TBST, then in TBS (without triton) and then coversliped with anti-fade medium. Images were taken on a Zeiss Z2 scanning microscope at x40 objective magnification, and show immunofluorescence labelling of SNAT localized to pinealocytes. Data courtesy of Dr. James Nagy's laboratory.
|Tested species reactivity||Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C(195) H T F L R R N S G C(205)|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||5-10 µg/ml|
|Western Blot (WB)||0.5 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-762 has successfully been used to detect serotonin N-AT in both WB and IF applications.
PA1-762 detects serotonin N-acetyltransferase (AA-NAT) from rat tissues.
PA1-762 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~23 kDa protein representing unphosphorylated and phosphorylated AA-NAT from rat pineal gland whole protein extract.
PA1-762 immunizing phosphopeptide (Cat. # PEP-066) corresponds to carboxy-terminal amino acid residues 198-208 of rat AA-NAT. Total anti-carboxy-terminal AA-NAT-specific IgG (Cat. # PA1-762) was affinity purified on resin-coupled non-phosphopeptide (Cat. # PEP-065). Phospho-specific carboxy-terminal IgG (Cat. # PA1-763) was subsequently isolated on resin-coupled phosphopeptide (Cat. # PEP-066). This sequence is completely conserved between rat and mouse AA-NAT protein.
Pineal gland serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT) is an ~23 kDa protein which is the rate-limiting enzyme in melatonin synthesis. Melatonin serves as the hormonal signal of the daily light/dark cycle, or body clock. The nocturnal oscillation in enzyme activity ranges by 10- to 100-fold depending on species among vertebrates. Studies have shown that AA-NAT is regulated by cAMP at both the mRNA and protein levels. AA-NAT has putative protein kinase A (PKA) sites at Thr29 and Ser206. Phosphorylation of AA-NAT at these residues might play a role in protein inactivation via proteasome-dependent degradation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.