Human FOXO3 (Forkhead Box Protein O3) ELISA Kit (HUES02194)

SKU:
HUES02194
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O43524
Sensitivity:
0.19ng/mL
Range:
0.31-20ng/mL
ELISA Type:
Sandwich
Reactivity:
Human
Tested Sample Types:
Serum, plasma and other biological fluids
$647

Description

ELISA Kit Technical ManualMSDS

Human FOXO3 (Forkhead Box Protein O3) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human FOXO3 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human FOXO3 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human FOXO3, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of Human FOXO3. The concentration of Human FOXO3 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human FOXO3 in samples. No significant cross-reactivity or interference between Human FOXO3 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(ng/mL)
O.D Average Corrected
202.44
2.49
2.4652.395
101.569
1.607
1.5881.518
50.972
0.966
0.9690.899
2.50.448
0.47
0.4590.389
1.250.243
0.243
0.2430.173
0.630.174
0.168
0.1710.101
0.310.113
0.129
0.1210.051
00.062
0.078
0.07--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FOXO3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FOXO3 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.992.556.690.912.316.81
Standard deviation0.050.150.320.050.130.21
C V (%)5.055.884.785.495.633.08

Recovery

The recovery of Human FOXO3 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)88-10394
EDTA plasma (n=5)87-10293
Cell culture media (n=5)85-10192

Linearity

Samples were spiked with high concentrations of Human FOXO3 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)95-11186-9989-102
Average (%)1019196
1:4Range (%)86-10180-9385-98
Average (%)928692
1:8Range (%)91-10582-9487-99
Average (%)968992
1:16Range (%)87-10186-9781-92
Average (%)939287

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human FOXO3 (Forkhead Box Protein O3) ELISA Kit (HUES02194) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37 °C.
    7. Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human FOXO3 (Forkhead Box Protein O3) ELISA Kit (HUES02194) Protein Information

UniProt Protein Function:FOXO3A: a transcription factors of the forkhead family. Contains 1 fork-head domain. May regulate apoptosis through control of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia.
UniProt Protein Details:

Protein type:Oncoprotein; DNA-binding; Transcription factor; Autophagy

Chromosomal Location of Human Ortholog: 6q21

Cellular Component: nucleoplasm; membrane; cytoplasm; cytosol; nucleus

Molecular Function:protein binding; DNA binding; chromatin DNA binding; sequence-specific DNA binding; transcription factor activity; protein kinase binding

Biological Process: epidermal growth factor receptor signaling pathway; transcription from RNA polymerase II promoter; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; DNA damage response, signal transduction by p53 class mediator; nerve growth factor receptor signaling pathway; initiation of primordial ovarian follicle growth; positive regulation of transcription, DNA-dependent; antral ovarian follicle growth; positive regulation of erythrocyte differentiation; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; ovulation from ovarian follicle; oocyte maturation; regulation of translation; regulation of transcription from RNA polymerase II promoter; positive regulation of neuron apoptosis; innate immune response; positive regulation of transcription from RNA polymerase II promoter

NCBI Summary:This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukemia. Alternatively spliced transcript variants encoding the same protein have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:O43524
NCBI GenInfo Identifier:8134467
NCBI Gene ID:2309
NCBI Accession:O43524.1
UniProt Secondary Accession:O43524,O15171, Q5T2I7, Q9BZ04, B4DVZ6, E1P5E6,
UniProt Related Accession:O43524
Molecular Weight:
NCBI Full Name:Forkhead box protein O3
NCBI Synonym Full Names:forkhead box O3
NCBI Official Symbol:FOXO3
NCBI Official Synonym Symbols:FOXO2; AF6q21; FKHRL1; FOXO3A; FKHRL1P2
NCBI Protein Information:forkhead box protein O3; forkhead box O3A; forkhead in rhabdomyosarcoma-like 1; forkhead homolog (rhabdomyosarcoma) like 1; forkhead, Drosophila, homolog of, in rhabdomyosarcoma-like 1
UniProt Protein Name:Forkhead box protein O3
UniProt Synonym Protein Names:AF6q21 protein; Forkhead in rhabdomyosarcoma-like 1
Protein Family:Forkhead box protein
UniProt Gene Name:FOXO3
UniProt Entry Name:FOXO3_HUMAN
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