Product Details
abID | ab65348 |
---|---|
Cat # +Size | K337-100 |
Size | 100 assays |
Detection Method | Absorbance (450 nm) |
Species Reactivity | Mammalian |
Applications | BioVision’s NADH/NAD Quantification Kit provides a convenient tool for sensitive detection of the intracellular nucleotides: NADH, NAD and their ratio. |
Features & Benefits | • Simple procedure; takes ~2 hours • Fast and convenient • Kit contains the necessary reagents for accurate measurement of NAD and NADH and their ratio. |
Kit Components | • NADH/NAD Extraction Buffer • NAD Cycling Buffer • NAD Cycling Enzyme Mix • NADH Developer • Stop Solution • NADH Standard (MW:763) |
Storage Conditions | -20°C |
Shipping Conditions | Gel Pack |
USAGE | For Research Use Only! Not For Use in Humans. |
Details
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The lower limit of detection is calculated by taking 20 pmole (lowest conc of Standard) divided by 50 µl (max sample volume) =20 pmol/50µl = 0.4 pmol/µl = 0.4 µM
The highest limit of detection is calculated by taking 100 pmol (highest conc of Standard) divided by 5 µl (min sample volume) = 100 pmol/5 µl = 20 pmol/µl = 20 µM
Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.
Harder A, Wildgruber R, Nawrocki A, Fey SJ, Larsen PM, Gorg A.
Source
Technical University of Munich, Department of Food Technology, Freising-Weihenstephan, Germany.
Abstract
Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii).
Your signals from 1 to 4 hrs of final incubation will ideally increase. Within that time range, whenever you are comfortable and satisfied with the signals, you can add the stop solution to terminate any more colour development.
For serum samples, although NADH consuming enzymes like LDH are present only in minute quantities, we suggest removing any enzyme that may consume NADH by filtering the serum through a 10 kDa spin filter (BV Cat# 1997) and using the flow through for the assay. Please do a pilot study to assess how much serum you need for the assay. You can try different sample volumes to ensure the readings are within the linear range of the standard curve.
You can measure two things with this assay, NADt, which is NAD+NADH and for this, you can directly use the filtered serum.
For measuring NADH, you need to decompose the existing NAD. Please place the serum in a tube and heat it at 60°C for 25-30 min. If serum proteins precipitate, centrifuge and take the supernatant for the assay. Heating will decompose all NAD and the serum will now have only NADH. Serum is now ready to be used for measuring NADH.