Human CASP14 (Caspase 14) CLIA Kit
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human CASP14 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CASP14 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 CASP14, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human CASP14. The concentration of Human CASP14 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||62.50-4000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human CASP14 in samples. No significant cross-reactivity or interference between Human CASP14 and analogues was observed.
As the RLU 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 CASP14 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CASP14 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||12.13||8.74||8.81||10.07||10.85||7.05|
The recovery of Human CASP14 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)||85-99||91|
|Cell culture media (n=5)||95-110||101|
Samples were spiked with high concentrations of Human CASP14 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)|
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.
|Micro CLIA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 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 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 (25X)||1 vial, 30 mL|
|Substrate Reagent A||1 vial, 5 mL||4'C (shading light)|
|Substrate Reagent B||1 vial, 5 mL||4'C (shading light)|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Human CASP14 (Caspase 14) CLIA Kit (HUES00434) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- 11. Determine the RLU value of each well immediately.
Human CASP14 (Caspase 14) CLIA Kit (HUES00434) Protein Information
|UniProt Protein Function:||CASP14: Believed to be a non-apoptotic caspase which is involved in epidermal differentiation. Seems to play a role in keratinocyte differentiation and cornification. Probably regulates maturation of the epidermis by proteolytically processing filaggrin. Complex of unprocessed caspase-14 and processed 19 kDa (p19) and 10 kDa (p10) subunits. In undifferentiated keratinocytes under postconfluency growth conditions (in vitro). Expressed in keratinocytes of adult skin suprabasal layers (from spinous layers to the stratum granulosum and stratum corneum). Expressed in keratinocytes of hair shaft and sebaceous glands. In psoriatic skin only expressed at very low levels. Belongs to the peptidase C14A family.|
|UniProt Protein Details:|
Protein type:EC 3.4.22.-; Protease
Chromosomal Location of Human Ortholog: 19p13.1
Cellular Component: cytoplasm; keratin filament; nucleus
Molecular Function:cysteine-type endopeptidase activity
Biological Process: epidermis development; DNA damage response, signal transduction resulting in induction of apoptosis; keratinization; proteolysis
|NCBI Summary:||This gene encodes a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This caspase has been shown to be processed and activated by caspase 8 and caspase 10 in vitro, and by anti-Fas agonist antibody or TNF-related apoptosis inducing ligand in vivo. The expression and processing of this caspase may be involved in keratinocyte terminal differentiation, which is important for the formation of the skin barrier. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||12231007|
|NCBI Gene ID:||23581|
|UniProt Secondary Accession:||P31944,O95823, Q3SYC9,|
|UniProt Related Accession:||P31944|
|Molecular Weight:||27,680 Da|
|NCBI Full Name:||Caspase-14|
|NCBI Synonym Full Names:||caspase 14, apoptosis-related cysteine peptidase|
|NCBI Official Symbol:||CASP14|
|NCBI Protein Information:||caspase-14; CASP-14; apoptosis-related cysteine protease; caspase 14, apoptosis-related cysteine protease|
|UniProt Protein Name:||Caspase-14|
|UniProt Gene Name:||CASP14|
|UniProt Entry Name:||CASPE_HUMAN|