|Detection Range:||62.50-4000 pg/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Human DAP6 in samples. No significant cross-reactivity or interference between Human DAP6 and analogues was observed.|
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 DAP6. 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 DAP6 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 DAP6, 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 DAP6. The concentration of Human DAP6 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||DAXX: a transcriptional co-regulatory protein. Proposed to mediate activation of the JNK pathway and apoptosis via ASK1 in response to signaling from FAS and TGF-betaR2. Glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, relocalizing Daxx from the nucleus to the cytoplasm, where Daxx binds to ASK1, and subsequently leads to ASK1 oligomerization. Interaction with HSP27 may prevent interaction with TGF-betaR2 and ASK1 and block DAXX-mediated apoptosis. Seems to act as a transcriptional co- repressor and inhibits PAX3 and ETS1 through direct protein- protein interaction. Its transcription repressor activity is modulated by recruiting it to subnuclear compartments like the nucleolus or PML/POD/ND10 nuclear bodies through interactions with MCSR1 and PML, respectively. Two alternatively spliced isoforms have been described.|
|UniProt Protein Details:|
Protein type:Nucleolus; Nuclear receptor co-regulator; Transcription, coactivator/corepressor; Apoptosis
Chromosomal Location of Human Ortholog: 6p21. 3
Cellular Component: chromosome, pericentric region; cytoplasm; cytosol; nucleoplasm; nucleus; PML body
Molecular Function:androgen receptor binding; enzyme binding; heat shock protein binding; histone binding; p53 binding; protein binding; protein kinase activator activity; protein kinase binding; protein N-terminus binding; receptor signaling protein activity; transcription corepressor activity; ubiquitin protein ligase binding
Biological Process: activation of JNK activity; androgen receptor signaling pathway; apoptosis; chromatin remodeling; induction of apoptosis via death domain receptors; negative regulation of transcription, DNA-dependent; nucleosome assembly; positive regulation of protein amino acid phosphorylation; positive regulation of protein kinase activity; regulation of protein ubiquitination; regulation of transcription, DNA-dependent
|NCBI Summary:||This gene encodes a multifunctional protein that resides in multiple locations in the nucleus and in the cytoplasm. It interacts with a wide variety of proteins, such as apoptosis antigen Fas, centromere protein C, and transcription factor erythroblastosis virus E26 oncogene homolog 1. In the nucleus, the encoded protein functions as a potent transcription repressor that binds to sumoylated transcription factors. Its repression can be relieved by the sequestration of this protein into promyelocytic leukemia nuclear bodies or nucleoli. This protein also associates with centromeres in G2 phase. In the cytoplasm, the encoded protein may function to regulate apoptosis. The subcellular localization and function of this protein are modulated by post-translational modifications, including sumoylation, phosphorylation and polyubiquitination. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2008]|
|NCBI GenInfo Identifier:||24636785|
|NCBI Gene ID:||1616|
|NCBI Accession:||Q9UER7. 2|
|UniProt Secondary Accession:||Q9UER7,O14747, O15141, O15208, Q5STK9, Q9BWI3, B4E1I3 F5ANJ6, F5ANJ7, F5H082,|
|UniProt Related Accession:||Q9UER7|
|Molecular Weight:||75,563 Da|
|NCBI Full Name:||Death domain-associated protein 6|
|NCBI Synonym Full Names:||death domain associated protein|
|NCBI Official Symbol:||DAXX|
|NCBI Official Synonym Symbols:||DAP6; EAP1; BING2|
|NCBI Protein Information:||death domain-associated protein 6|
|UniProt Protein Name:||Death domain-associated protein 6|
|UniProt Synonym Protein Names:||Daxx; hDaxx; ETS1-associated protein 1; EAP1; Fas death domain-associated protein|
|Protein Family:||Death domain-associated protein|
|UniProt Gene Name:||DAXX|
|UniProt Entry Name:||DAXX_HUMAN|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human DAP6 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human DAP6 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.67||5.41||3.40||6.33||6.00||4.51|
The recovery of Human DAP6 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||89-103||96|
|Cell culture media (n=5)||91-102||97|
Samples were spiked with high concentrations of Human DAP6 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)|
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.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-20°C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4°C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4°C(shading light)|
|Stop Solution||1 vial, 10 mL||4°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.