Human UBE1L (Ubiquitin Activating Enzyme E1 Like Protein) ELISA Kit (HUES02317)
Human UBE1L (Ubiquitin Activating Enzyme E1 Like Protein) 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 UBE1L . 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 UBE1L 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 UBE1L, 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 UBE1L. The concentration of Human UBE1L in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||1.56-100 mIU/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Human UBE1L in samples. No significant cross-reactivity or interference between Human UBE1L and analogues was observed.
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human UBE1L were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human UBE1L were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.79||4.63||4.03||5.58||5.56||3.83|
The recovery of Human UBE1L 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)||87-102||93|
|Cell culture media (n=5)||84-99||91|
Samples were spiked with high concentrations of Human UBE1L 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 ELISA 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||1 vial, 10 mL||4'C(shading light)|
|Stop Solution||1 vial, 10 mL||4'C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Human UBE1L (Ubiquitin Activating Enzyme E1 Like Protein) ELISA Kit (HUES02317) 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 UBE1L (Ubiquitin Activating Enzyme E1 Like Protein) ELISA Kit (HUES02317) Protein Information
|UniProt Protein Function:||UBE1L: Activates ubiquitin by first adenylating with ATP its C- terminal glycine residue and thereafter linking this residue to the side chain of a cysteine residue in E1, yielding an ubiquitin- E1 thioester and free AMP. Belongs to the ubiquitin-activating E1 family.|
|UniProt Protein Details:|
Protein type:Ubiquitin conjugating system; Ubiquitin ligase; EC 22.214.171.124
Chromosomal Location of Human Ortholog: 3p21
Cellular Component: cytosol; nucleoplasm; nucleus
Molecular Function:ISG15 activating enzyme activity; protein binding; ubiquitin activating enzyme activity; ubiquitin-protein ligase activity
Biological Process: bypass DNA synthesis; ISG15-protein conjugation; modification-dependent protein catabolic process; negative regulation of interferon type I production; protein modification process
|NCBI Summary:||The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. This gene encodes a member of the E1 ubiquitin-activating enzyme family. The encoded enzyme is a retinoid target that triggers promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha) degradation and apoptosis in acute promyelocytic leukemia, where it is involved in the conjugation of the ubiquitin-like interferon-stimulated gene 15 protein. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||215273977|
|NCBI Gene ID:||7318|
|UniProt Secondary Accession:||P41226,Q9BRB2,|
|UniProt Related Accession:||P41226|
|Molecular Weight:||111,694 Da|
|NCBI Full Name:||Ubiquitin-like modifier-activating enzyme 7|
|NCBI Synonym Full Names:||ubiquitin like modifier activating enzyme 7|
|NCBI Official Symbol:||UBA7|
|NCBI Official Synonym Symbols:||D8; UBE2; UBA1B; UBE1L|
|NCBI Protein Information:||ubiquitin-like modifier-activating enzyme 7|
|UniProt Protein Name:||Ubiquitin-like modifier-activating enzyme 7|
|UniProt Synonym Protein Names:||D8; Ubiquitin-activating enzyme E1 homolog|
|Protein Family:||Ubiquitin-like modifier-activating enzyme|
|UniProt Gene Name:||UBA7|
|UniProt Entry Name:||UBA7_HUMAN|