Human USP8 (Ubiquitin Specific Peptidase 8) ELISA Kit (HUES03259)

SKU:
HUES03259
Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
€539

Description

ELISA Kit Technical ManualMSDS

Human USP8 (Ubiquitin Specific Peptidase 8) 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 USP8 . 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 USP8 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 USP8, 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 USP8. The concentration of Human USP8 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 Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human USP8 in samples. No significant cross-reactivity or interference between Human USP8 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
(ng/mL)
O.D Average Corrected
202.49
2.51
2.52.417
101.595
1.609
1.6021.519
50.914
0.88
0.8970.814
2.50.465
0.487
0.4760.393
1.250.247
0.243
0.2450.162
0.630.184
0.18
0.1820.099
0.310.133
0.133
0.1330.05
00.077
0.089
0.083--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human USP8 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human USP8 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.962.319.900.902.139.64
Standard deviation0.060.130.400.050.120.43
C V (%)6.255.634.045.565.634.46

Recovery

The recovery of Human USP8 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)92-110100
EDTA plasma (n=5)89-10295
Cell culture media (n=5)92-10799

Linearity

Samples were spiked with high concentrations of Human USP8 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 (%)95-10994-11087-99
Average (%)10310193
1:4Range (%)92-10983-9681-96
Average (%)998888
1:8Range (%)88-9983-9480-91
Average (%)938887
1:16Range (%)87-10386-10081-91
Average (%)949287

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 USP8 (Ubiquitin Specific Peptidase 8) ELISA Kit (HUES03259) 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 USP8 (Ubiquitin Specific Peptidase 8) ELISA Kit (HUES03259) Protein Information

UniProt Protein Function:USP8: Hydrolase that can remove conjugated ubiquitin from proteins and therefore plays an important regulatory role at the level of protein turnover by preventing degradation. Converts both 'Lys-48' an 'Lys-63'-linked ubiquitin chains. Catalytic activity is enhanced in the M phase. Involved in cell proliferation. Required to enter into S phase in response to serum stimulation. May regulate T-cell anergy mediated by RNF128 via the formation of a complex containing RNF128 and OTUB1. Probably regulates the stability of STAM2 and RASGRF1. Regulates endosomal ubiquitin dynamics, cargo sorting, membrane traffic at early endosomes, and maintenance of ESCRT-0 stability. The level of protein ubiquitination on endosomes is essential for maintaining the morphology of the organelle. Deubiquitinates EPS15 and controles tyrosine kinase stability. Removes conjugated ubiquitin from EGFR thus regulating EGFR degradation and downstream MAPK signaling. Involved in acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and microtubules. Deubiquitinates BIRC6/bruce and KIF23/MKLP1. Forms a ternary complex with RNF128 and OTUB1. Interacts (via C-terminal UCH catalytic domain) with OTUB1 isoform 1. Interacts with STAM2 (via SH3 domain). Interacts with DNAJB3, EGFR, EPS15, RASGRF1, RNF41, YWHAE, YWHAG and YWHAZ. Interacts with NBR1, RASGRF1, RNF41 and IST1. Associates with the ESCRT-0 complex and with microtubules. Interacts with BIRC6/bruce and KIF23/MKLP1. Upon growth stimulation in starved human fibroblasts. Decreases in response to growth arrest induced by cell-cell contact. Belongs to the peptidase C19 family.
UniProt Protein Details:

Protein type:Ubiquitin-specific protease; EC 3.4.19.12; Ubiquitin conjugating system; Protease

Chromosomal Location of Human Ortholog: 15q21.2

Cellular Component: extrinsic to plasma membrane; nucleoplasm; Golgi apparatus; early endosome; cytoplasm; acrosome; midbody; cytosol

Molecular Function:protein binding; cysteine-type endopeptidase activity; ubiquitin-specific protease activity; SH3 domain binding

Biological Process: proteasomal ubiquitin-dependent protein catabolic process; cell proliferation; protein deubiquitination; endosome organization and biogenesis; cytokinesis after mitosis; Ras protein signal transduction

NCBI Summary:This gene encodes a protein that belongs to the ubiquitin-specific processing protease family of proteins. The encoded protein is thought to regulate the morphology of the endosome by ubiquitination of proteins on this organelle and is involved in cargo sorting and membrane trafficking at the early endosome stage. This protein is required for the cell to enter the S phase of the cell cycle and also functions as a positive regulator in the Hedgehog signaling pathway in development. Pseudogenes of this gene are present on chromosomes 2 and 6. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Sep 2013]
UniProt Code:P40818
NCBI GenInfo Identifier:731046
NCBI Gene ID:9101
NCBI Accession:P40818.1
UniProt Secondary Accession:P40818,Q2TB31, Q7Z3U2, Q86VA0, Q8IWI7, B4DKA8,
UniProt Related Accession:P40818
Molecular Weight:1118
NCBI Full Name:Ubiquitin carboxyl-terminal hydrolase 8
NCBI Synonym Full Names:ubiquitin specific peptidase 8
NCBI Official Symbol:USP8
NCBI Official Synonym Symbols:UBPY; SPG59; HumORF8
NCBI Protein Information:ubiquitin carboxyl-terminal hydrolase 8; ubiquitin isopeptidase Y; deubiquitinating enzyme 8; ubiquitin thiolesterase 8; ubiquitin-specific-processing protease 8
UniProt Protein Name:Ubiquitin carboxyl-terminal hydrolase 8
UniProt Synonym Protein Names:Deubiquitinating enzyme 8; Ubiquitin isopeptidase Y; hUBPy; Ubiquitin thioesterase 8; Ubiquitin-specific-processing protease 8
UniProt Gene Name:USP8
UniProt Entry Name:UBP8_HUMAN
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