FTO ELISA Kit (Fat Mass and Obesity-Associated)

Details for Product FTO ELISA Kit No. ABIN956444, Anbieter: Anmelden zum Anzeigen
Antigen
  • AW743446
  • mKIAA1752
  • RGD1305121
  • FTO, alpha-ketoglutarate dependent dioxygenase
  • fat mass and obesity associated
  • fat mass and obesity associated L homeolog
  • FTO
  • Fto
  • fto.L
Epitop
Intracellular
Reaktivität
Human
Alternativen
Kits mit alternativen Reaktivitäten:
4
3
2
Methodentyp
Sandwich ELISA
Detektionsbereich
0.156-10 ng/mL
Untere Nachweisgrenze
0.156 ng/mL
Applikation
ELISA
Optionen
Hersteller
Anmelden zum Anzeigen
Hersteller Produkt- Nr.
Anmelden zum Anzeigen
Proben Cell Lysate
Analytische Methode Quantitative
Nachweismethode Colorimetric
Spezifität This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human FTO in cells. A monoclonal antibody specific for FTO has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, FTO is recognized by the addition of a purified polyclonal antibody specific for FTO (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of FTO in the samples. This ELISA is specific for the measurement of natural and recombinant human FTO. It does cross-react with human adiponectin, human RBP4, human Nampt, human vaspin, human progranulin, human resistin, human clusterin, human GPX3, human sirtuin 1, human IDO, human IL-33, human ANGPTL3, human ANGPTL4, human FGF21, mouse progranulin, mouse ANGPTL3, mouse leptin, rat Nampt. The assay range is 0.156 - 10 ng FTO/mL and a detection limit of 50 pg/mL (based on adding two standard deviations to the mean value of the (50) zero standards).
Produktmerkmale FTO, Fat mass-and obesity-associated gene, was discovered as a responsible gene causing the mouse 'fused toes' mutation. The predicted 502-amino acid Fto protein has a calculated molecular mass of 58 kD and contains an N-terminal bipartite nuclear localization signal. FTO is widely expressed in a variety of human tissues, with highest levels in brain and pancreatic islets. Bioinformatics analysis indicates that FTO shares sequence motifs with iron- and 2-oxoglutarate (2OG)-dependent oxygenases. The FTO (human) (IntraCellular) ELISA Kit is to be used for the in vitro quantitative determination of human FTO in cell lysates or cell-based assays (screening). This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human FTO in cells. A monoclonal antibody specific for FTO has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, FTO is recognized by the addition of a purified polyclonal antibody specific for FTO (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of FTO in the samples. This ELISA is specific for the measurement of natural and recombinant human FTO. It does cross-react with human adiponectin, human RBP4, human Nampt, human vaspin, human progranulin, human resistin, human clusterin, human GPX3, human sirtuin 1, human IDO, human IL-33, human ANGPTL3, human ANGPTL4, human FGF21, mouse progranulin, mouse ANGPTL3, mouse leptin, rat Nampt. The assay range is 0.156 - 10 ng FTO/mL and a detection limit of 50 pg/mL (based on adding two standard deviations to the mean value of the (50) zero standards).

Features and Benefits:
  • Simple procedure
  • Fast and convenient
  • High-throughput adaptable
Bestandteile
  • Pre-coated Microtiter Plate
  • Wash Buffer (10X)
  • Diluent (5X)
  • Lysis Buffer (10X)
  • Detection Antibody
  • Detector 100X (Hrp conjugated anti-IgG)
  • Human FTO Standard (lyophilized, 20 ng)
  • Human FTO QC Sample (lyophilized)
  • TMB Substrate Solution
  • Stop Solution
  • Plate Sealers
Andere Bezeichnung FTO (FTO ELISA Kit Abstract)
Forschungsgebiet Cardiovascular, Atherosclerosis, Metabolism
Applikationshinweise This assay is to be used for the in vitro quantitative determination of human FTO in the range of 0.156 - 10 ng FTO/mL and a detection limit of 50 pg/mL (based on adding two standard deviations to the mean value of the (50) zero standards).
Kommentare

Absorbance (450 nm)
Simple procedure
Fast and convenient
High-throughput adaptable

Plattentyp Pre-coated
Protokoll a) Determine the number of 8-well strips needed for assay and insert them into the frame for current use. The extra strips should be resealed in the foil pouch and can be stored at 4 °C for up to 1 month.
b) Add 100 l of the Standards, Samples and QC Sample into the appropriate wells in duplicate.
c) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
d) Aspirate and wash x 3 with 300 l of 1X Wash Buffer.
e) Add 100 l Detection Antibody to each well and tap gently on the side of the plate to mix.
f) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
g) Aspirate and wash x 3 with 300 l of 1X Wash Buffer.
h) Add 100 l of the 1X Detector to each well.
i) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
j) Remove plate from 37 °C, aspirate and wash x 5 with 300 l of 1X Wash Buffer.
k) After last wash, tap inverted plate on a stack of paper towels. Complete removal of liquid is essential for good performance.
l) Add 100 l of the TMB Substrate Solution to each well.
m) Allow the color to develop at room temperature in the dark for 10 min.
n) Stop the reaction by adding 100 l of Stop Solution to each well.
o) Tap the plate gently to ensure thorough mixing. The substrate reaction yields a blue solution that turns yellow when Stop Solution is added. Caution: Stop Solution is a Corrosive Solution p) Measure the OD at 450 nm in an ELISA plate reader within 30 min.
Aufbereitung der Reagenzien

Prepare just the appropriate amounts for the assay.
a) 1X Wash Buffer: Dilute 10X Wash Buffer 1: 9 with dH2O to obtain 1X Wash Buffer.
b) 1X Diluent: Dilute 5X Wash Buffer 1: 4 with dH2O to obtain 1X Diluent.
c) 1X Lysis Buffer: Dilute 10X Lysis Buffer 1: 9 with dH2O to obtain 1X Lysis Buffer.
d) 1X Detector: Dilute 100X Detector 1: 99 with 1X Diluent to obtain 1X Detector.
e) Detection Antibody & TMB Substrate Solution: Ready to use. Warm to room temp before use. Note: The diluted Detector must be used within 1 hr of preparation.

Testdurchführung

Test Samples/Standards/QC Sample: (We recommend these be run in duplicate)
a) Cell Lysates: Grow cells to 90 % confluency. Scrap cells off the plate and transfer to an appropriate tube. Keep on ice and microcentrifuge at 1,200 rpm for 5 min at 4 °C. Remove supernatant, rinse cells once with ice-cold PBS. Remove PBS and add 200 µL ice-cold 1X Lysis Buffer supplemented with 1 mM phenyl methylsulfonyl fluoride (PMSF) to ten million cells. Incubate on ice for 30 min. Microcentrifuge at 12,000 rpm for 5 min at 4 °C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Use freshly prepared cell lysate samples. Note: Cell lysates have to be diluted in Diluent 1X. Samples containing visible precipitates must be clarified before use. As starting point 1/10 to 1/1000 dilutions are recommended.
b) QC Sample: Reconstitute human FTO QC Sample with 1 mL of dH2O. Mix the QC Sample to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The QC Sample is ready to use-do not dilute it (refer to the C of A for current QC Sample concentration).
c) Standards: Reconstitute human FTO Standard with 1 mL of dH2O to produce a stock solution (20 ng/mL). Mix the Stock solution to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The reconstituted standard should be aliquoted and stored at -20 °C.
d) Prepare Standard Curve using 2-fold serial dilutions with 1X Diluent.

Ergebnisberechnung

a) Average the duplicate readings for each Standard, QC Sample and Test Sample and subtract the average blank value (obtained with the 0 ng/mL point).
b) Generate a Standard Curve by plotting the average absorbance on the horizontal (X) axis vs. the corresponding concentration (µg /mL) on the vertical (Y) axis.
c) Calculate the Test Sample FTO concentrations by interpolation of the Standard Curve regression curve in the form of a quadratic equation.
d) If the Test Samples were diluted, multiply the interpolated values by the dilution factor to calculate the corrected human FTO concentrations.

Testpräzision 1. Intra-assay Precision: (4) samples of known concentrations of human FTO were assayed in replicates (6) times to test precision within an assay.
2. Inter-assay Precision: (3) samples of known concentrations of human FTO were assayed in (4) separate assays to test precision between assays.
3. Recovery: Different human cell lysates were spiked with known concentrations of human FTO and the recoveries averaged 100 % (range from 95 % to 105 %).
Beschränkungen Nur für Forschungszwecke einsetzbar
Lagerung 4 °C
Informationen zur Lagerung Reagents must be stored at 2 - 8 °C when not in use. Bring reagents to room temperature before use. Do not expose reagents to temperatures greater than 25 °C.
Haltbarkeit 12 months
Bilder des Herstellers
ELISA image for Fat Mass and Obesity-Associated (FTO) ELISA Kit (ABIN956444) Fat Mass and Obesity-Associated (FTO) ELISA Kit
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