Human UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) 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 UCHL1 . 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 UCHL1 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 UCHL1, 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 UCHL1. The concentration of Human UCHL1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||15.63-1000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human UCHL1 in samples. No significant cross-reactivity or interference between Human UCHL1 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 UCHL1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human UCHL1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||12.97||8.60||6.68||12.15||11.78||6.62|
The recovery of Human UCHL1 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)||98-114||107|
|Cell culture media (n=5)||90-101||96|
Samples were spiked with high concentrations of Human UCHL1 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 UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) CLIA Kit (HUES01226) 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 100 µL 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 UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) CLIA Kit (HUES01226) Protein Information
|UniProt Protein Function:||UCHL1: Ubiquitin-protein hydrolase involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. This enzyme is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin. Also binds to free monoubiquitin and may prevent its degradation in lysosomes. The homodimer may have ATP-independent ubiquitin ligase activity. Monomer. Homodimer. Interacts with SNCA. Interacts with COPS5. Found in neuronal cell bodies and processes throughout the neocortex. Expressed in neurons and cells of the diffuse neuroendocrine system and their tumors. Weakly expressed in ovary. Down-regulated in brains from Parkinson disease and Alzheimer disease patients. Belongs to the peptidase C12 family.|
|UniProt Protein Details:|
Protein type:Ubiquitin conjugating system; Cell development/differentiation; Ligase; Protease; EC 18.104.22.168
Chromosomal Location of Human Ortholog: 4p14
Cellular Component: nucleoplasm; endoplasmic reticulum membrane; cell soma; axon; cytoplasm; plasma membrane; cytosol
Molecular Function:ubiquitin binding; omega peptidase activity; protein binding; cysteine-type endopeptidase activity; ubiquitin-specific protease activity; alpha-2A adrenergic receptor binding; ligase activity
Biological Process: cell proliferation; proteasomal ubiquitin-dependent protein catabolic process; negative regulation of MAP kinase activity; axon transport of mitochondrion; protein deubiquitination; axon target recognition; eating behavior; neuromuscular process; muscle fiber development; sensory perception of pain; adult walking behavior
Disease: Neurodegeneration With Optic Atrophy, Childhood-onset; Parkinson Disease 5, Autosomal Dominant
|NCBI Summary:||The protein encoded by this gene belongs to the peptidase C12 family. This enzyme is a thiol protease that hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin. This gene is specifically expressed in the neurons and in cells of the diffuse neuroendocrine system. Mutations in this gene may be associated with Parkinson disease.[provided by RefSeq, Sep 2009]|
|NCBI GenInfo Identifier:||136681|
|NCBI Gene ID:||7345|
|UniProt Secondary Accession:||P09936,Q4W5K6, Q71UM0,|
|UniProt Related Accession:||P09936|
|NCBI Full Name:||Ubiquitin carboxyl-terminal hydrolase isozyme L1|
|NCBI Synonym Full Names:||ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)|
|NCBI Official Symbol:||UCHL1|
|NCBI Official Synonym Symbols:||NDGOA; PARK5; PGP95; PGP9.5; Uch-L1; HEL-117; PGP 9.5|
|NCBI Protein Information:||ubiquitin carboxyl-terminal hydrolase isozyme L1; ubiquitin thioesterase L1; epididymis luminal protein 117; neuron cytoplasmic protein 9.5; ubiquitin C-terminal hydrolase|
|UniProt Protein Name:||Ubiquitin carboxyl-terminal hydrolase isozyme L1|
|UniProt Synonym Protein Names:||Neuron cytoplasmic protein 9.5; PGP 9.5; PGP9.5; Ubiquitin thioesterase L1|
|Protein Family:||Ubiquitin carboxyl-terminal hydrolase|
|UniProt Gene Name:||UCHL1|
|UniProt Entry Name:||UCHL1_HUMAN|