Nadp nadph detection kit

Manufactured by Beyotime

Sourced in China

The NADP+/NADPH detection kit is a laboratory tool designed to quantify the levels of NADP+ and NADPH in biological samples. This kit provides a colorimetric assay for the direct measurement of these important cellular metabolites.

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7 protocols using nadp nadph detection kit

Intracellular NADPH Quantification

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About 1 × 10⁶ cells were collected and washed with PBS buffer three times, then ground with liquid nitrogen to break the cell walls. Intracellular NADPH content was determined using the NADP⁺/NADPH Detection Kit of Beyotime Biotechnology Co., Ltd., Shanghai, China.

Liu B., Sun X., Liu Y., Yang M., Wang L., Li Y, & Wang J. (2022). Increased NADPH Supply Enhances Glycolysis Metabolic Flux and L-methionine Production in Corynebacterium glutamicum. Foods, 11(7), 1031.

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NADPH Quantification Protocol

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NADPH levels were determined using a NADP+/NADPH Detection Kit (Beyotime, Shanghai, China). Tissue samples of 10 to 30 mg were homogenized on ice with 400 μL NADP+/NADPH-extracting solution, then centrifuged at 12,000× g for 10 min at 4 °C and 50 μL supernatants were used to measure NADPH. After being heated in a water bath at 60 °C for 30 min, each supernatant sample was added into 100 μL G6PDH working liquid in a 96-well plate and incubated at 37 °C for 10 min. Following this, color reaction was performed for 30 min. Finally, each plate was measured at 450 nm for the absorbance.

Cai S., Duo T., Wang X., Tong X., Luo C., Chen Y., Li J, & Mo D. (2022). A Comparative Analysis of Metabolic Profiles of Embryonic Skeletal Muscle from Lantang and Landrace Pigs. Animals : an Open Access Journal from MDPI, 12(4), 420.

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Antibody and Reagent Utilization Protocol

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Antibodies and reagents used in the study are as follows: anti‐TSP50 antibody was prepared and purified by Abcam (1:1000). Anti‐G6PD (25413‐1‐AP, 1:1000), anti‐GST (10000‐0‐AP, 1:1000), anti‐Flag (20543‐1‐AP, 1:1000), anti‐SIRT2 (19655‐1‐AP, 1:1000), anti‐pan acetylation antibody (66289‐1‐Ig, 1:1000), anti‐O‐glycosylation antibody (20415‐1‐AP, 1:1000), rabbit IgG, anti‐actin antibody (60008‐1‐Ig, 1:5000) and mouse IgG were purchased from Protein‐tech Group. Anti‐G6PD K171ac was designed and prepared by Absin Bioscience Inc (1:500), and anti‐KAT9 was purchased from Bioss Antibodies (bs‐20539R, 1:500). NADP+ was purchased from MedChemExpress. NADP+/NADPH detection kit and G6PD activity detection kit were purchased from Beyotime. TG and T‐CHO detection kit were purchased from Nanjing Jiancheng Bioengineering Institute. A glutaraldehyde solution was purchased from Sigma‐Aldrich.

Zhang X., Gao F., Ai H., Wang S., Song Z., Zheng L., Wang G., Sun Y, & Bao Y. (2021). TSP50 promotes hepatocyte proliferation and tumour formation by activating glucose‐6‐phosphate dehydrogenase (G6PD). Cell Proliferation, 54(4), e13015.

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NADPH Production Quantification

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The NADPH production was measured using the NADP+/NADPH detection Kit (Beyotime Technology) according to the manufacture’s protocol.

Zhang R., Tian Z., Xu Y, & Lv L. (2024). D-mannose promotes the degradation of IDH2 through upregulation of RNF185 and suppresses breast cancer. Nutrition & Metabolism, 21, 5.

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Glucose and Lactate Metabolism Assays

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Processed cells and cell culture media were collected according to the protocol. Glucose levels in the culture medium were determined using the Glucose Uptake Colorimetric Assay Kit (BioVision). Lactate levels were measured using a Lactate Colorimetric Assay Kit (BioVision). Intracellular G6P level and G6PDH enzyme activity were determined using a G6P Assay Kit (Beyotime) or G6PDH Activity Assay Kit (Beyotime), respectively. In addition, the NADP⁺/NADPH detection kit (Beyotime) detected the NADP⁺/NADPH ratio.

Liang J., Liu Q., Xia L., Lin J., Oyang L., Tan S., Peng Q., Jiang X., Xu X., Wu N., Tang Y., Su M., Luo X., Yang Y., Liao Q, & Zhou Y. (2022). Rac1 promotes the reprogramming of glucose metabolism and the growth of colon cancer cells through upregulating SOX9. Cancer Science, 114(3), 822-836.

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NADP/NADPH and ATP Quantification

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The cells in the six-well plate were collected into EP tube, and the contents of total NADP (NADPH and NADP⁺) in the cells were determined according to the methodology of NADP⁺/NADPH detection kit (S0179, Beyotime, China). The concentration of NADP⁺ was calculated as follows: [NADP⁺] = [NADP_total] - [NADPH]. The ATP detection was performed according to protocol of ATP assay kit (A095-1-1, Nanjing Jiancheng Bioengineering Institute, China). All experiments were performed in triplicate and measured accurately with unit protein amount.

Fang Y., Dou R., Huang S., Han L., Fu H., Yang C., Song J., Zheng J., Zhang X., Liu K., Xiang Z., Zhang X., Wang S, & Xiong B. (2022). LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis. International Journal of Biological Sciences, 18(7), 3082-3101.

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Comprehensive Analysis of Mitochondrial Function

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Step RT-qPCR Kit (TIANGEN) was used to detect MCU mRNA expression, and Western blot was used to detect MCU protein expression. JC-1 Mitochondrial membrane potential detection kit (Beyotime) detects mitochondrial membrane potential, ATP detection kit (Beyotime) detects cardiomyocyte mitochondrial ATP activity, mitochondrial calcium dye Rhod-2 (Abcam) was used to detect myocardial cell mitochondrial calcium uptake capacity, and Mito-SOX reagent (Thermo Fisher Scientific) was used to detect Changes of reactive oxygen species (ROS) in cardiac mitochondria. NADP + /NADPH detection kit (Beyotime) and GSH/GSSG detection kit (Beyotime) were used to detect myocardial cells NADP + /NADPH, GSH/GSSG. Western blot was used to detect the expression of caspase-3 (Abcam), cleared caspase-9 (Abcam) and Bcl-2 (Abcam). Flow cytometry combined with Annexin V-FITC/PI staining (BD) and TUNEL apoptosis detection kit (RIBO) was used to detect cardiomyocyte apoptosis.

Chen X., Guo W., Jing Z., Zhang T., Wu Z., Cui H., Zheng W., Chen Y., & Chen H. (2020). The mitochondrial calcium uniporter induces apoptosis in cardiomyocytes cultured with high-glucose medium by affecting mitochondrial function.

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