UBD ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Ubiquitin D (UBD) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Ubiquitin D (UBD) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Ubiquitin-like protein modifier which can be covalently attached to target protein and subsequently leads to their degradation by the 26S proteasome, in a NUB1-dependent manner. Probably functions as a survival factor. Conjugation ability activated by UBA6. Promotes the expression of the proteasome subunit beta type-9 (PSMB9/LMP2) . Regulates TNF-alpha-induced and LPS-mediated activation of the central mediator of innate immunity NF-kappa-B by promoting TNF-alpha-mediated proteasomal degradation of ubiquitinated-I-kappa-B-alpha. Required for TNF-alpha-induced p65 nuclear translocation in renal tubular epithelial cells (RTECs) . May be involved in dendritic cell (DC) maturation, the process by which immature dendritic cells differentiate into fully competent antigen-presenting cells that initiate T-cell responses. Mediates mitotic non-disjunction and chromosome instability, in long-term in vitro culture and cancers, by abbreviating mitotic phase and impairing the kinetochore localization of MAD2L1 during the prometaphase stage of the cell cycle. May be involved in the formation of aggresomes when proteasome is saturated or impaired. Mediates apoptosis in a caspase-dependent manner, especially in renal epithelium and tubular cells during renal diseases such as polycystic kidney disease and Human immunodeficiency virus (HIV) -associated nephropathy (HIVAN) .

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Ubiquitin D (UBD) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Ubiquitin D (UBD) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Ubiquitin D (UBD), 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 Ubiquitin D (UBD) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery range (%) Average (%) Serum (n=5) 78-98 91 EDTA Plasma (n=5) 93-103 101 Heparin Plasma (n=5) 79-91 84 Matrix Recovery range (%) Average (%) Matrix Recovery range (%) Average (%) Serum (n=5) 78-98 91 Serum (n=5) 78-98 91 EDTA Plasma (n=5) 93-103 101 EDTA Plasma (n=5) 93-103 101 Heparin Plasma (n=5) 79-91 84 Heparin Plasma (n=5) 79-91 84

Component Amount Anti-UBD Microplate 96 Wells (12 x 8 Well strips) UBD Lyophilized Standard 2 100X Biotinylated UBD Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL Component Amount Component Amount Anti-UBD Microplate 96 Wells (12 x 8 Well strips) Anti-UBD Microplate 96 Wells (12 x 8 Well strips) UBD Lyophilized Standard 2 UBD Lyophilized Standard 2 100X Biotinylated UBD Detector Antibody 1 x 120 uL 100X Biotinylated UBD Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL

This assay has high sensitivity and excellent specificity for detection of Ubiquitin D (UBD) . No significant cross-reactivity or interference between Ubiquitin D (UBD) and analogues was observed.