Human Cell Biology ELISA Kits 4
Human UBE3A (Ubiquitin Protein Ligase E3A) CLIA Kit (HUES00717)
- SKU:
- HUES00717
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 37.5pg/mL
- Range:
- 62.5-4000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human UBE3A in samples. No significant cross-reactivity or interference between Human UBE3A 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 UBE3A. 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 UBE3A 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 UBE3A, 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 UBE3A. The concentration of Human UBE3A in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and transfers it to its substrates. Several substrates have been identified including the RAD23A and RAD23B, MCM7 (which is involved in DNA replication), annexin A1, the PML tumor suppressor, and the cell cycle regulator CDKN1B. Catalyzes the high-risk human papilloma virus E6-mediated ubiquitination of p53/TP53, contributing to the neoplastic progression of cells infected by these viruses. Additionally, may function as a cellular quality control ubiquitin ligase by helping the degradation of the cytoplasmic misfolded proteins. Finally, UBE3A also promotes its own degradation in vivo. Plays an important role in the regulation of the circadian clock: involved in the ubiquitination of the core clock component ARNTL/BMAL1, leading to its proteasomal degradation (PubMed:24728990). |
| NCBI Summary: | This gene encodes an E3 ubiquitin-protein ligase, part of the ubiquitin protein degradation system. This imprinted gene is maternally expressed in brain and biallelically expressed in other tissues. Maternally inherited deletion of this gene causes Angelman Syndrome, characterized by severe motor and intellectual retardation, ataxia, hypotonia, epilepsy, absence of speech, and characteristic facies. The protein also interacts with the E6 protein of human papillomavirus types 16 and 18, resulting in ubiquitination and proteolysis of tumor protein p53. Alternative splicing of this gene results in three transcript variants encoding three isoforms with different N-termini. Additional transcript variants have been described, but their full length nature has not been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q05086 |
| NCBI GenInfo Identifier: | 215274240 |
| NCBI Gene ID: | 7337 |
| NCBI Accession: | Q05086. 4 |
| UniProt Secondary Accession: | Q05086,P78355, Q93066, Q9UEP4, Q9UEP5, Q9UEP6, Q9UEP7 Q9UEP8, Q9UEP9, A8K8Z9, |
| UniProt Related Accession: | Q05086 |
| Molecular Weight: | 100,102 Da |
| NCBI Full Name: | Ubiquitin-protein ligase E3A |
| NCBI Synonym Full Names: | ubiquitin protein ligase E3A |
| NCBI Official Symbol: | UBE3A |
| NCBI Official Synonym Symbols: | AS; ANCR; E6-AP; HPVE6A; EPVE6AP |
| NCBI Protein Information: | ubiquitin-protein ligase E3A |
| UniProt Protein Name: | Ubiquitin-protein ligase E3A |
| UniProt Synonym Protein Names: | E6AP ubiquitin-protein ligase; HECT-type ubiquitin transferase E3A; Human papillomavirus E6-associated protein; Oncogenic protein-associated protein E6-AP; Renal carcinoma antigen NY-REN-54 |
| UniProt Gene Name: | UBE3A |
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.
| Concentration (pg/mL) | RLU | Average | Corrected |
| 4000 | 54240 63922 | 59081 | 59046 |
| 2000 | 25942 29776 | 27859 | 27824 |
| 1000 | 13536 13432 | 13484 | 13449 |
| 500 | 6187 7023 | 6605 | 6570 |
| 250 | 3314 3172 | 3243 | 3208 |
| 125 | 1618 1544 | 1581 | 1546 |
| 62.50 | 741 769 | 755 | 720 |
| 0 | 34 36 | 35 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human UBE3A were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human UBE3A were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 186.53 | 343.31 | 1638.06 | 198.04 | 310.29 | 1602.45 |
| Standard deviation | 18.26 | 24.07 | 167.90 | 20.30 | 32.08 | 177.07 |
| C V (%) | 9.79 | 7.01 | 10.25 | 10.25 | 10.34 | 11.05 |
Recovery
The recovery of Human UBE3A 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) | 91-106 | 99 |
| EDTA plasma (n=5) | 83-99 | 90 |
| Cell culture media (n=5) | 96-113 | 103 |
Linearity
Samples were spiked with high concentrations of Human UBE3A 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) | ||
| 1:2 | Range (%) | 103-116 | 99-114 | 99-114 |
| Average (%) | 110 | 106 | 105 | |
| 1:4 | Range (%) | 92-106 | 99-112 | 99-111 |
| Average (%) | 99 | 106 | 106 | |
| 1:8 | Range (%) | 92-104 | 102-118 | 95-111 |
| Average (%) | 97 | 109 | 103 | |
| 1:16 | Range (%) | 83-97 | 87-98 | 93-105 |
| Average (%) | 90 | 93 | 99 |
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 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 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl 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 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.
- 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.
- Add 100µL 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 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.
- Determine the RLU value of each well immediately.
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