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Nitric oxide synthase (neuronal) (human), (recombinant) (purified)

 
ALX-201-261-C010 10 µg 401.00 USD
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Product Specification

Alternative Name:nNOS, NOS I
 
MW:~160kDa/subunit; homodimer.
 
Source:Produced in E. coli.
 
EC:1.14.13.39
 
UniProt ID:P29475
 
Concentration:~1mg/ml
 
Formulation:Liquid. In 40mM bis-tris-propane, pH 7.8, containing 150mM sodium chloride, 0.1% BSA, 5µM tetrahydrobiopterin, 0.5mM DTT and 5% glycerol.
 
Purity:≥90% (SDS-PAGE and heme spectral analysis)
 
Specific Activity:≥400-600nmol/min/mg (U/mg) protein. Amount assayed 0.5-1µg. Optimal conditions: 37°C, 40mM TRIS-HCl, HEPES or EPPS buffer, pH 7.6, 10µM oxyhemoglobin, 0.3mM DTT, 1mM NADPH, 4µM FAD, 4µM FMN, 4µM H4B, 100units/ml catalase, 10units/ml superoxide dismutase, 0.1mg/ml BSA, 35µl of 9mM EDTA, 35µl of 150mg/ml calmodulin and 12.5mM CaCl2 in a final volume of 1ml.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Use/Stability:Stable for at least 1 year after receipt when stored at -80°C. Keep stock vial on ice at all times!
 
Handling:Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -80°C.
 
Protocol:Preparation of oxyhemoglobin:
1) Wash a G-50 column with your assay buffer of your choice.
2) Weigh out 2-3mg hemoglobin in eppendorf tube and add 200µl of your column buffer.
3) Add several crystals of dithionite to reduce hemoglobin to oxyhemoglobin.
4) Drip onto G-50 column.
5) Wash with 500µl buffer; repeat a couple of times.
6) Collect brownish eluate in eppendorf tube and store on ice.
7) Wash column with water and reuse again.
8) Use ~10-15µl of oxyhemoglobin in the assay corresponding to ~10µM.
 

General Literature References

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli: L.J. Roman, et al.; PNAS 92, 8428 (1995), Abstract; Full Text
Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer: H.M. Abu-Soud and D.J. Stuehr; PNAS 90, 10769 (1993), Abstract; Full Text

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