Human FOXO4 (Forkhead Box Protein O4) ELISA Kit (HUES02195)

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ELISA Kit Technical ManualMSDS

Human FOXO4 (Forkhead Box Protein O4) 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 FOXO4 . 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 FOXO4 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 FOXO4, 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 FOXO4. The concentration of Human FOXO4 in samples can be calculated by comparing the OD of the samples to the standard curve.

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


This kit recognizes Human FOXO4 in samples. No significant cross-reactivity or interference between Human FOXO4 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.

O.D Average Corrected


Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FOXO4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FOXO4 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Mean (ng/mL)0.981.819.051.001.728.38
Standard deviation0.050.100.390.070.070.27
C V (%)5.105.524.317.004.073.22


The recovery of Human FOXO4 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)85-10092
EDTA plasma (n=5)94-105100
Cell culture media (n=5)86-9991


Samples were spiked with high concentrations of Human FOXO4 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 (%)95-11191-10694-107
Average (%)10297100
1:4Range (%)89-10186-9990-101
Average (%)959295
1:8Range (%)91-10785-9988-102
Average (%)989294
1:16Range (%)94-10880-9387-97
Average (%)1008592

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 FOXO4 (Forkhead Box Protein O4) ELISA Kit (HUES02195) 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 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl 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 100µL 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 350µL 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 100µL 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 90µL 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 FOXO4 (Forkhead Box Protein O4) ELISA Kit (HUES02195) Protein Information

UniProt Protein Function:FOXO4: transcription factor AFX1 containing 1 fork-head domain. May play a role in the insulin signaling pathway. Involved in acute leukemias by a chromosomal translocation t(X;11)(q13;q23) that involves MLLT7 and MLL/HRX. The result is a rogue activator protein. Two splice-variant isoforms have been described.
UniProt Protein Details:

Protein type:Oncoprotein; Transcription factor; DNA-binding

Chromosomal Location of Human Ortholog: Xq13.1

Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus

Molecular Function:beta-catenin binding; DNA binding; enzyme binding; identical protein binding; protein binding; sequence-specific DNA binding; transcription factor activity; transcription factor binding

Biological Process: cell cycle arrest; insulin receptor signaling pathway; negative regulation of angiogenesis; negative regulation of cell proliferation; negative regulation of smooth muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; stem cell differentiation; transcription from RNA polymerase II promoter

NCBI Summary:This gene encodes a member of the O class of winged helix/forkhead transcription factor family. Proteins encoded by this class are regulated by factors involved in growth and differentiation indicating they play a role in these processes. A translocation involving this gene on chromosome X and the homolog of the Drosophila trithorax gene, encoding a DNA binding protein, located on chromosome 11 is associated with leukemia. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2010]
UniProt Code:P98177
NCBI GenInfo Identifier:110825720
NCBI Gene ID:4303
NCBI Accession:P98177.5
UniProt Secondary Accession:P98177,O43821, Q13720, Q3KPF1, Q8TDK9, B7WPJ7,
UniProt Related Accession:P98177
Molecular Weight:47,941 Da
NCBI Full Name:Forkhead box protein O4
NCBI Synonym Full Names:forkhead box O4
NCBI Official Symbol:FOXO4  
NCBI Official Synonym Symbols:AFX; AFX1; MLLT7  
NCBI Protein Information:forkhead box protein O4
UniProt Protein Name:Forkhead box protein O4
UniProt Synonym Protein Names:Fork head domain transcription factor AFX1
Protein Family:Forkhead box protein
UniProt Gene Name:FOXO4  
UniProt Entry Name:FOXO4_HUMAN
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Additional Information

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