Human USP8 (Ubiquitin Specific Peptidase 8) CLIA Kit (HUES01236)
- Product type:
- ELISA Kit
- ELISA Type:
|Detection range:||62.50-4000 pg/mL|
|Sample type:||Serum, plasma and other biological fluids|
|Repeatability:||CV < 15%|
|Specificity:||This kit recognizes Human USP8 in samples. No significant cross-reactivity or interference between Human USP8 and analogues was observed.|
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 USP8. 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 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 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 USP8. The concentration of Human USP8 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|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]|
|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|
|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|
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 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|
|C V (%)||8.03||9.42||7.49||8.28||10.00||7.59|
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 (%)|
|EDTA plasma (n=5)||87-100||93|
|Cell culture media (n=5)||98-115||105|
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)|
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 ×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 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|
- 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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 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.
- 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.
- Add 100 µL 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 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.
- Determine the RLU value of each well immediately.