Background[1-3]
The human nicotinamide phosphoribosyltransferase (NAMPT) ELISA kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) to detect the in vitro concentration of human nicotinamide phosphoribosyltransferase (NAMPT). To the coated microwells pre-coated with human nicotinamide phosphoribosyltransferase (NAMPT) capture antibody, add the specimen, standard, and HRP-labeled detection antibody in sequence, incubate and wash thoroughly. The substrate TMB is used to develop color. TMB is converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of the color is positively correlated with nicotinamide phosphoribosyltransferase (NAMPT) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and calculate the sample concentration.
Nicotinamide phosphoribosyltransferase (NAMPT) is the key rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthetic pathway, also known as visfatin (Visfatin) or pre-B cell clone enhancer (PBEF). It affects many processes such as metabolism, inflammatory response, cell proliferation, differentiation and apoptosis, especially aging, by regulating the NAD levels of the body or cells and through other non-enzymatic mechanisms.
Experimental operation:
1. Dilution and addition of standards
2. Add samples: Set up blank wells (no samples and enzyme-labeled reagents are added to the blank control wells, and the remaining steps are the same) and sample wells to be tested. First add 40 μl of sample diluent to the well of the sample to be tested on the enzyme-labeled coated plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme plate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: Seal the plate with sealing film and incubate at 37°C for 30 minutes.
4. Solution preparation: Dilute 30 times (20 times of 48T) concentrated washing liquid with distilled water 30 times (20 times of 48T) and set aside.
5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme label reagent to each well, except blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: First add 50μl of chromogen A to each well, then add 50μl of chromogen B, shake gently to mix evenly, and develop color for 15 minutes at 37°C in the dark.
10. Termination: Add 50 μl of stop solution to each well to terminate the reaction (blue turns to yellow immediately).
11. Measurement: Measure the absorbance (OD value) of each well sequentially with a blank air conditioner zero and a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.
Apply[4][5]
Study on the relationship between NAMPT and NAPRT, the key enzymes used in the NAD salvage synthesis pathway, and the prognosis of colorectal cancer patients
Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT) are key enzymes in the NAD salvage biosynthetic pathway. They synthesize NAD with each other, thus providing the main source of NAD biosynthesis. However, the prognostic value of NAMPT and NAPRT in colorectal cancer (CRC) is unclear.
Methods: Clinical cases were collected, and clinical case data were obtained through 5-year follow-up. Postoperative tissues from 261 patients were collected to make tissue chips, and immunohistochemistry was used to study the expression of NAMPT and NAPRT proteins in CRC cancer tissues and adjacent tissues.
Apply bioinformatics techniques and methods to collect public database information, and use online analysis software such as UALCAN, PROGgnesV2, cBioportal, and TargetScan to analyze the expression, mutation, amplification, promoter methylation, etc. of NAPRT/NAMPT in CRC. , and its impact on the prognosis of CRC. KEGG pathway analysis was performed on NAPRT/NAMPT-related genes.
Cell experiments, PCR, WesternBlot, CCK-8, Tanswell and other methods were used to study the function of Clariant pigment NAMPT in CRC in vitro. As a result, we found that NAPRT and NAMPT are highly expressed in CRC tissues, and the high expression of NAPRT or NAMPT is related to vascular invasion, invasion depth and advanced TNM stage in CRC.
High expression of NAPRT or NAMPT predicts shorter overall survival and disease-free survival time in CRC patients. In addition, the expression of NAMPT and NAPRT in CRC tissues is positively correlated. Patients with NAPRThigh/NAMPThigh had the shortest survival time.
References
[1]Nicotinamide phosphoribosyl transferase regulates cell growth via the Sirt1/P53 signaling pathway and is a prognosis marker in colorectal cancer[J].Jing-hua Pan,Hong Zhou,Sheng-Bin Zhu,Jin-Lian Huang,Xiao ‐Xu Zhao,Hui Ding,Li Qin,Yun‐Long Pan.Journal of Cellular Physiology.2019(4)
[2]microRNA-300/NAMPT regulates inflammatory responses through activation of AMPK/mTOR signaling pathway in neonatal sepsis[J].Yexuzi Li,Junzhong Ke,Chen Peng,Fugen Wu,Yukang Song.Biomedicine&Pharmacotherapy.2018
[3]NAD metabolism: Implications in aging and longevity[J].Keisuke Yaku,Keisuke Okabe,Takashi Nakagawa.Ageing Research Reviews.2018
[4]Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].Freddie Bray BSc,MSc,PhD,Jacques Ferlay ME,Isabelle Soerjomataram MD,MSc,PhD,Rebecca L .Siegel MPH,Lindsey A.Torre MSPH,Ahmedin Jemal PhD,DVM.CA:A Cancer Journal for Clinicians.2018(6)
[5]Li Xiaoqin. Study on the relationship between NAMPT and NAPRT, the key enzymes of the NAD salvage synthesis pathway, and the prognosis of colorectal cancer patients[D]. Chongqing Medical University, 2020.
