NAMPT ELISA Kit

Produktnummer ABIN1979293

Produktdetails NAMPT ELISA Kit

Target: NAMPT (Nicotinamide phosphoribosyltransferase (NAMPT))

Reaktivität: Human, Maus, Ratte

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Detektionsbereich: 0.1-1.000 ng/mL

Untere Nachweisgrenze: 0.1 ng/mL

Applikation: ELISA

Verwendungszweck: Human/Mouse/Rat Visfatin EIA Kit optimized for serum and cell culture medium. Competition-based ELISA on a 96-well strip plate.

Proben: Cell Culture Supernatant, Serum

Analytische Methode: Quantitative

Spezifität: Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC.

Kreuzreaktivität (Details): This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC.

Sensitivität: 0.778 ng/mL

Produktmerkmale:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Bestandteile:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Standard Peptide
• Assay Diluent(s)
• Biotinylated Peptide
• HRP-Streptavidin
• TMB One-Step Substrate
• Stop Solution
• Assay Diagram
• Positive Control Sample
• Capture Antibody
• User Manual

Benötigtes Material:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Orbital shaker
• Aluminum foil
• Saran Wrap
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• SigmaPlot software (or other software that can perform four-parameter logistic regression models)

Anwendungsinformationen

Applikationshinweise: Recommended Dilution for serum and plasma samplesHuman: 4X / Mouse: 4X / Rat: 2X

Probenmenge: 100 μL

Testdauer: 5 h

Plattentyp: Pre-coated

Protokoll:

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 μL detection antibody to each well.
3. Incubate 1.5 h at RT or O/N at 4 °C.
4. Add 100 μL standard or sample to each well.
5. Incubate 2.5 h at RT.
6. Add 100 μL prepared streptavidin solution.
7. Incubate 45 min at RT.
8. Add 100 μL TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL Stop Solution to each well.
11. Read plate at 450 nm immediately.

Aufbereitung der Reagenzien:

1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
   2. Briefly centrifuge the Visfatin Antibody vial (Item N) and reconstitute with 5 µL of ddH2O before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
   3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-Visfatin antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
   4. Briefly centrifuge the vial of biotinylated Visfatin peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL 1x Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated Visfatin will be 10 ng/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
   5. Preparation of Standards: Label 6 microtubes with the following concentrations: 1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 100 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated Visfatin solution into each tube, except for the 1000 ng/mL (leave this one empty). It is very important to make sure the concentration of biotinylated Visfatin is 10 ng/mL in all standards. a. Briefly centrifuge the vial of standard Visfatin peptide (Item C) and reconstitute with 10 µL of ddH2O. In the tube labeled 1000 ng/mL, pipette 8 µL of Item C and 792 µL of 10 ng/mL biotinylated Visfatin solution (prepared in step 4 above). This is your Visfatin stock solution (1000 ng/mL Visfatin, 10 ng/mL biotinylated Visfatin). Mix thoroughly. This solution serves as the first standard. b. To make the 100 ng/mL standard, pipette 50 µL of Visfatin stock solution the tube labeled 100 ng/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated Visfatin and 50 mL of the prior concentration until 100 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL Visfatin, 10 ng/mL biotinylated Visfatin) serves as the zero standard (or total binding).
   6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the 1X Assay Diluent E. This solution will be used in steps 7 and
   9.
   7. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 2 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated Visfatin is 10 ng/mL.
   8. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer.
   9. Sample Preparation: Use 1X Assay Diluent E + biotinylated Visfatin to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Visfatin is 10 ng/mL in every sample.
   Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Visfatin to a final concentration of 10 ng/mL.
   Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
   10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 100- fold with 1X Assay Diluent E.

Aufbereitung der Proben:

Use 1X Assay Diluent E + biotinylated Visfatin to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Visfatin is 10 ng/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Visfatin to a final concentration of 10 ng/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.

Testdurchführung:

1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL anti-Visfatin antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C. 0
   3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each), Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
   5. Discard the solution and wash 4 times as directed in Step
   3.
   6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes with gentle shaking at room temperature. It is recommended that incubation time should not be shorter or longer than 45 minutes.
   7. Discard the solution and wash 4 times as directed in Step
   3.
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec).
   9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately. 1

Ergebnisberechnung:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

Testpräzision: Intra-Assay: CV < 10 %
Inter-Assay: CV < 15 %

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid repeated freeze/thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: Standard, Biotinylated Visfatin peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.

Haltbarkeit: 6 months

Referenzen für NAMPT ELISA Kit (ABIN1979293)

Pisani, Dumortier, Beranger, Casamento, Ghandour, Giroud, Gautier, Balaguer, Chambard, Virtanen, Nuutila, Niemi, Taittonen, Van Obberghen, Hinault, Amri: "Visfatin expression analysis in association with recruitment and activation of human and rodent brown and brite adipocytes." in: Adipocyte, Vol. 5, Issue 2, pp. 186-95, (2016) (PubMed).

Gümü?, Güzel, Topcu, Timur, Y?lmaz, Dan??man: "Plasma Visfatin Levels in Adolescents with Polycystic Ovary Syndrome: A Prospective Case-Control Study." in: Journal of pediatric and adolescent gynecology, Vol. 28, Issue 4, pp. 249-53, (2015) (PubMed).

Luizon, Belo, Palei, Amaral, Lacchini, Sandrim, Duarte, Cavalli, Tanus-Santos: "Effects of NAMPT polymorphisms and haplotypes on circulating visfatin/NAMPT levels in hypertensive disorders of pregnancy." in: Hypertension research : official journal of the Japanese Society of Hypertension, Vol. 38, Issue 5, pp. 361-6, (2015) (PubMed).

Wu, Chen, Wei, Luo, Yan: "Effect of non-surgical periodontal treatment on visfatin concentrations in serum and gingival crevicular fluid of patients with chronic periodontitis and type 2 diabetes mellitus." in: Journal of periodontology, Vol. 86, Issue 6, pp. 795-800, (2015) (PubMed).

Souza-Smith, Siggins, Molina: "Mesenteric Lymphatic-Perilymphatic Adipose Crosstalk: Role in Alcohol-Induced Perilymphatic Adipose Tissue Inflammation." in: Alcoholism, clinical and experimental research, Vol. 39, Issue 8, pp. 1380-7, (2015) (PubMed).

Rodrigues, Pietrani, Bosco, Ferreira, Sandrim, Gomes: "Visfatin levels are decreased in advanced stages of diabetic nephropathy." in: Renal failure, Vol. 37, Issue 9, pp. 1529-30, (2015) (PubMed).

Zhou, Pan, Huang, Jia, Zhao, Chen, Diao, Wan, Cui: "Visfatin induces cholesterol accumulation in macrophages through up-regulation of scavenger receptor-A and CD36." in: Cell stress & chaperones, Vol. 18, Issue 5, pp. 643-52, (2014) (PubMed).

Guzel, Seven, Guzel, Buyuk, Celebi, Aydemir: "Visfatin, Leptin, and TNF-?: Interrelated Adipokines in Insulin-Resistant Clinical and Subclinical Hypothyroidism." in: Endocrine research, (2013) (PubMed).

Mabrouk, Ghareeb, Shehab, Omar, El-Kabarity, Soliman, Mohamed: "Serum visfatin, resistin and IL-18 in A group of Egyptian obese diabetic and non diabetic individuals." in: The Egyptian journal of immunology / Egyptian Association of Immunologists, Vol. 20, Issue 1, pp. 1-11, (2013) (PubMed).

Yanni, Darendeliler, Bas, Kucukemre Aydin, Coban, Ince: "The role of leptin, soluble leptin receptor, adiponectin and visfatin in insulin sensitivity in preterm born children in prepubertal ages." in: Cytokine, Vol. 64, Issue 1, pp. 448-53, (2013) (PubMed).

Güdücü, İşçi, Görmüş, Yiğiter, Dünder: "Serum visfatin levels in women with polycystic ovary syndrome." in: Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology, Vol. 28, Issue 8, pp. 619-23, (2012) (PubMed).

Karakan, Sezer, Özdemir Acar, Haberal: "The relationship of visfatin levels with insulin resistance and left ventricular hypertrophy in peritoneal dialysis patients." in: Renal failure, Vol. 34, Issue 6, pp. 732-7, (2012) (PubMed).

Kim, Lee, Kim, Park, Kim, Kang, Lee, Kim, Kim, Suh, Eom: "Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866." in: Journal of molecular biology, Vol. 362, Issue 1, pp. 66-77, (2006) (PubMed).

Hug, Lodish: "Medicine. Visfatin: a new adipokine." in: Science (New York, N.Y.), Vol. 307, Issue 5708, pp. 366-7, (2005) (PubMed).

Kralisch, Klein, Bluher, Paschke, Stumvoll, Fasshauer: "Therapeutic perspectives of adipocytokines." in: Expert opinion on pharmacotherapy, Vol. 6, Issue 6, pp. 863-72, (2005) (PubMed).

Fukuhara, Matsuda, Nishizawa, Segawa, Tanaka, Kishimoto, Matsuki, Murakami, Ichisaka, Murakami, Watanabe, Takagi, Akiyoshi, Ohtsubo, Kihara, Yamashita, Makishima, Funahashi, Yamanaka, Hiramatsu et al.: "Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. ..." in: Science (New York, N.Y.), Vol. 307, Issue 5708, pp. 426-30, (2005) (PubMed).

Revollo, Grimm, Imai: "The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells." in: The Journal of biological chemistry, Vol. 279, Issue 49, pp. 50754-63, (2004) (PubMed).

Levin, Kennedy: "Operations improvement. Opportunities for group practices. Part II." in: Group practice journal, Vol. 38, Issue 3, pp. 39-48, (1989) (PubMed).

Trautmann, Kearns: "Diabetes and the eye." in: Postgraduate medicine, Vol. 45, Issue 5, pp. 133-9 (PubMed).

Details zu NAMPT

Target: NAMPT (Nicotinamide phosphoribosyltransferase (NAMPT))

Andere Bezeichnung: Visfatin (NAMPT Produkte)

Hintergrund: Visfatin, Nicotinamide phosphoribosyltransferase (Nampt), pre-B-cell colony-enhancing factor 1 (PBEF)

Gen-ID: 10135

UniProt: P43490