Rat E3 ubiquitin-protein ligase ELISA Kit

From Assay Genie, this Sandwich ELISA kit (double-antibody format) is intended for the quantitative measurement of E3 ubiquitin-protein ligase in rat samples using a colorimetric detection method. The kit is suitable for serum, plasma, cell culture supernatants, cell or tissue lysates and other liquid samples, and is positioned for cell biology research. According to the manufacturer, E3 ubiquitin protein ligases regulate the ubiquitination process involved in protein degradation and cellular control, and their dysregulation has been implicated in various diseases.

The assay reports a sensitivity of 0.469 ng/mL and an assay range of 0.781-50 ng/mL. Linearity was verified across dilution ratios of 1:2, 1:4 and 1:8 in serum (86-102%, 94-100%, 82-99%), EDTA plasma (92-104%, 87-98%, 80-93%) and heparin plasma (85-103%, 83-93%, 92-100%), each with n = 5. Spike-and-recovery testing yielded ranges of 85-105% (average 95%) for serum, 88-104% (average 94%) for EDTA plasma, and 86-102% (average 94%) for heparin plasma. The associated target protein carries the synonyms Huwe1, HECT, UBA and WWE domain-containing protein 1, Upstream regulatory element-binding protein 1, URE-B1 and URE-binding protein 1 (UniProt P51593). Unopened kits are stored at 2-8°C for 12 months.

The workflow equilibrates reagents and TMB substrate to room temperature, then loads standards, samples and blanks onto the pre-coated plate with a 37°C, 90-minute primary incubation. A biotin-labelled detection antibody is added (37°C, 60 minutes), followed by HRP-Streptavidin SABC (37°C, 30 minutes). TMB substrate is incubated in the dark for 10-20 minutes, stop solution is added, and absorbance is read at 450 nm. Serum is clotted and centrifuged at 1000 × g for 20 minutes; plasma is collected into anticoagulant tubes and centrifuged at 1000 × g for 15 minutes at 2-8°C; tissue homogenates, cell culture supernatants and cell lysates are prepared in buffers containing protease inhibitors. For additional sample-type guidance, the manufacturer's technical support can be contacted at [email protected].