Human UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) ELISA Kit (HUES03248)

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HUES03248
€539

Description

ELISA Kit Technical ManualMSDS

Human UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) 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 UCHL1 . 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 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 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 UCHL1. The concentration of Human UCHL1 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human UCHL1 in samples. No significant cross-reactivity or interference between Human UCHL1 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.

Concentration
(pg/mL)
O.D Average Corrected
50002.617
2.639
2.6282.548
25001.687
1.715
1.7011.621
12501.018
1.018
1.0180.938
6250.484
0.486
0.4850.405
312.50.323
0.301
0.3120.232
156.250.197
0.183
0.190.11
78.130.132
0.14
0.1360.056
00.073
0.087
0.08--

Precision

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
Sample123123
n202020202020
Mean (pg/mL)235.15513.922459.26238.21478.222622.50
Standard deviation16.2528.8386.0714.4121.9597.56
C V (%)6.915.613.506.054.593.72

Recovery

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 (%)
Serum (n=5)91-10397
EDTA plasma (n=5)91-10799
Cell culture media (n=5)90-10597

Linearity

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)
1:2Range (%)91-10693-11091-106
Average (%)9710097
1:4Range (%)88-10182-9485-97
Average (%)948790
1:8Range (%)88-10087-10189-101
Average (%)939294
1:16Range (%)88-10181-9089-100
Average (%)938694

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 UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) ELISA Kit (HUES03248) 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 UCHL1 (Ubiquitin Carboxyl Terminal Hydrolase L1) ELISA Kit (HUES03248) 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 3.4.19.12

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]
UniProt Code:P09936
NCBI GenInfo Identifier:136681
NCBI Gene ID:7345
NCBI Accession:P09936.2
UniProt Secondary Accession:P09936,Q4W5K6, Q71UM0,
UniProt Related Accession:P09936
Molecular Weight:223
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
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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