Sensitive Measurements of NADP+ and NADPH
使用该试剂盒中的提取缓冲液和过滤管，可以通过脱蛋白轻松制备来自细胞培养物的细胞裂解物。 细胞内 NADPH 水平可以通过细胞裂解物的热处理来量化。 此外，可以通过从独立测量的总 NADP+/NADPH 水平中减去 NADPH 水平来确定细胞内 NADP+ 水平。该试剂盒可用于测量多达 12 个样品和 8 个标准样品 (n=3)。 当测量超过 12 个样品时，需要准备额外的过滤管。
Study of NADP+/NADPH as Markers
NADP+ 和 NADPH 的细胞内水平被认为是了解癌细胞和线粒体功能如何受药物管理、基因工程等影响的重要代谢标志物。特别是，出版物数量有所增加，主要是在美国，这表明 细胞内代谢的评估被认为对了解细胞状态很重要。
Producing Accurate Data in Plate AssayThe quantitative analysis of NADPH and total NADP+/NADPH levels (0.01-1.00 μmol/l) is possible with the simultaneous measurement of the standard buffer supplied with this kit. When the total NADP+/NADPH levels in samples are higher than 1 μmol/l, the quantitative analysis becomes possible with the use of the diluted samples.It has been confirmed that the NADP/NADPH Assay Kit-WST reacts with neither NAD+ nor NADH (see right diagram below).
Example of Measurements in Combination with Glutathione (GSH) Quantification KitChange in metabolic activity was examined when anticancer drug doxorubicin (Dox) was added to Jurkat cells.Dox was added to Jurkat cells (3×106 cells) to obtain a final concentration of 500 nmol/l Dox, and after 24 hours incubation, NADP+/NADPH ratio and reduced/oxidized glutathione (GSH/GSSG) ratio were determined using the NADP/NADPH Assay Kit-WST and the GSSG/GSH Quantification Kit (Item#: G257), respectively, after the cells were washed in phosphate buffered saline (PBS).
The results shown above are likely to be explained by the following mechanism. When DOX (doxorubicin) was added to cells, DOX radicals, along with NADP+, were generated by enzymatic reaction. DOX radicals form reactive oxygen species (ROS), which induces DNA damage and apoptosis.In the meantime, to eliminate ROS formed in cells, GSH is consumed and GSSG is increased.Moreover, NADPH is used to reduce GSSG to GSH, resulting in an increase of NADP+.
* The number of samples that can be recorded when the sample is measured in triplicates.The number of samples that can be measured when the standard sample is serially diluted from 2 μmol / l is shown in the table above. Using the data collected, a calibration curve is created at a total of 8 points (n=3).It is necessary to create a calibration curve per measurement when you perform the measurement separately. If the measurement is done separately, the above sample number will be less.
No, we don’t sell the filtration tube included in the kit separately. If you need additional supplies, you can also use a commercially available filtration tube.Supplier：Pall CorporationProduct：Nanosep® MF Centrifugal Devices (MWCO：10K, color：blue)
You can’t store the Working solution. Please prepare the Working solution prior to use. Please protect from light because the Working solution ins light-sensitive. The Working solution is stable for 4 hours at room temperature with protection from light.