Human OCT4 (Octamer Binding Transcription Factor 4) ELISA Kit

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SKU:
HUFI01494
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q01860
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
OCT4, Octamer Binding Transcription Factor 4, POU5F1, POU domain, class 5, transcription factor 1, Octamer-binding transcription factor 3, OTF-3, Octamer-binding protein 3, Oct-3, Octamer-binding protein 4, Oct-4, OCT3, OTF3, OTF4
Reactivity:
Human
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Description

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Product Name:Human OCT4 (Octamer Binding Transcription Factor 4) ELISA Kit
Product Code:HUFI01494
Size:96 Assays
Alias:OCT4, Octamer Binding Transcription Factor 4, POU5F1, POU domain, class 5, transcription factor 1, Octamer-binding transcription factor 3, OTF-3, Octamer-binding protein 3, Oct-3, Octamer-binding protein 4, Oct-4, OCT3, OTF3, OTF4
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human POU5F1 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human POU5F1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human POU5F1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)96-10399
EDTA plasma(n=5)89-10495
UFH plasma(n=5)85-10293
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human POU5F1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)91-102%89-103%87-103%
EDTA plasma(n=5)84-99%84-95%83-93%
UFH plasma(n=5)82-98%85-99%85-94%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotQ01860
UniProt Protein Function:Oct4: Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Forms a trimeric complex with SOX2 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206. Critical for early embryogenesis and for embryonic stem cell pluripotency. Interacts with UBE2I and ZSCAN10. Interacts with PKM2. Interacts with WWP2. Transcriptional activity is positively regulated by PKM2. Expressed in developing brain. Highest levels found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. Low levels of expression in adult tissues. Belongs to the POU transcription factor family. Class- 5 subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 6p21.31

Cellular Component: nucleoplasm; transcription factor complex; cytoplasm; cytosol; nucleus

Molecular Function:protein binding; miRNA binding; DNA binding; sequence-specific DNA binding; ubiquitin protein ligase binding; transcription factor binding; transcription factor activity

Biological Process: transcription from RNA polymerase II promoter; blastocyst development; anatomical structure morphogenesis; regulation of asymmetric cell division; regulation of transcription, DNA-dependent; regulation of gene expression; somatic stem cell maintenance; endodermal cell fate specification; response to wounding; positive regulation of transcription from RNA polymerase II promoter; mRNA transcription from RNA polymerase II promoter

NCBI Summary:This gene encodes a transcription factor containing a POU homeodomain that plays a key role in embryonic development and stem cell pluripotency. Aberrant expression of this gene in adult tissues is associated with tumorigenesis. This gene can participate in a translocation with the Ewing's sarcoma gene on chromosome 21, which also leads to tumor formation. Alternative splicing, as well as usage of alternative AUG and non-AUG translation initiation codons, results in multiple isoforms. One of the AUG start codons is polymorphic in human populations. Related pseudogenes have been identified on chromosomes 1, 3, 8, 10, and 12. [provided by RefSeq, Oct 2013]
UniProt Code:Q01860
NCBI GenInfo Identifier:42560248
NCBI Gene ID:5460
NCBI Accession:NP_002692
UniProt Secondary Accession:Q01860,P31359, Q15167, Q15168, Q16422, Q5STF3, Q5STF4 A6NCS1, A6NLL8, D2IYK4,
UniProt Related Accession:Q01860
Molecular Weight:30,085 Da
NCBI Full Name:POU domain, class 5, transcription factor 1 isoform 1
NCBI Synonym Full Names:POU class 5 homeobox 1
NCBI Official Symbol:POU5F1
NCBI Official Synonym Symbols:OCT3; OCT4; OTF3; OTF4; OTF-3; Oct-3; Oct-4
NCBI Protein Information:POU domain, class 5, transcription factor 1; octamer-binding protein 3; octamer-binding protein 4; POU domain transcription factor OCT4; octamer-binding transcription factor 3; POU-type homeodomain-containing DNA-binding protein
UniProt Protein Name:POU domain, class 5, transcription factor 1
UniProt Synonym Protein Names:Octamer-binding protein 3; Oct-3; Octamer-binding protein 4; Oct-4; Octamer-binding transcription factor 3
UniProt Gene Name:POU5F1
UniProt Entry Name:PO5F1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
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