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Human E3 Ubiquitin-Protein Ligase TRIM21 (RO52) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to RO52. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to RO52. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain RO52, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of RO52 in the samples is then determined by comparing the O.D. of the samples to the standard curve.


For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B ('Thr-187' phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139, PubMed:16297862, PubMed:16316627, PubMed:16472766, PubMed:16880511, PubMed:18022694, PubMed:18361920, PubMed:18641315, PubMed:18845142, PubMed:19675099). Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through 'Lys-63'-linked ubiquitination of NMI (PubMed:26342464).

GENE ID 6737
SWISS PROT P19474
SYNONYMS 52 kDa ribonucleoprotein autoantigen Ro/SS-A; 52 kDa Ro protein; Ro(SS-A);RING finger protein 81; Tripartite motif-containing protein 21; Sjoegren syndrome type A antigen(SS-A)


Materials Supplied

Kit Components 96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate 1 plate
Plate sealer for 96 wells 2
Standard
2 tubes
Diluent buffer 1 bottle
Detection Reagent A 1 bottle
Detection Reagent B 1 bottle
TMB Substrate 1 tube
Stop Solution 1 tube
Wash Buffer (30 ℅ concentrate) 1 tube
Product data sheet 1 copy

Storage

Storage The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY

Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level RO52 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level RO52 were tested on 3 different plates, with 8 replicates in each plate.
CV(%) = SD/meanX100

Intra-Assay: CV<10%
Inter-Assay: CV<12%

SENSITIVITY The minimum detectable dose was 0.126ng/mL.
ASSAY RANGE 0.312-20ng/mL
SPECIFICITY This assay has high sensitivity and excellent specificity for the detection of RO52.
No significant cross-reactivity or interference between RO52 and analogs was observed.
Note:
Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between RO52 and all analogs, therefore, cross reactivity may still exist.