UCHL-1 Sandwich ELISA Kit

Ubiquitin C-terminal hydrolase 1 (UCHL1) was independently discovered by several different research groups and so has several other names, such as ubiquitin carboxyl esterase L1, ubiquitin thiolesterase, neuron-specific protein Pgp9.5 and Park5. It was originally identified as a major neuron specific cytoplasmic protein from 2dimensional gel analysis of brain tissues and immunostaining, and was given the name “protein gene product 9.5” or Pgp9.5 (1).

UCHL1 is the first characterized member of a family of ubiquitin Cterminal hydrolases with 4 members in the human. These enzymes cleave ubiquitin from other molecules, an activity important to generate mono-ubiquitin from ubiquitin genes which encode either polyubiquitin chains or ubiquitin fused to other proteins. Ubiquitin functions as a protein tag which covalently attaches to other proteins and targets them for proteosomal degradation. UCHL1 activity may also remove ubiquitin from partially degraded proteins, allowing the ubiquitin monomer to be recycled. Regulation of the ubiquitin pathway is very important and many disease states are associated with defects in this pathway.

The kit is an ELISA capture assay, in which a 96-well-format plate is coated with an affinity purified mouse monoclonal antibody raised against the purified recombinant human UCHL1. The plate has been blocked with a protein solution to remove any further non-specific binding, and is supplied with 50 µL of Tris buffered saline (TBS) plus 5 mM sodium azide (NaN3) preservative. Standards, quality controls and samples are incubated in microplate wells for 3 hours at room temperature or overnight at 4OC. Binding is detected with a rabbit polyclonal antibody to UCHL1 for another 3 hours. The amount of specifically bound rabbit polyclonal antibody is then detected using a goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP). Finally, the wells are incubated with 3, 3`, 5, 5`-Tetramethyl Benzidine (TMB), a reagent which produces a blue color when exposed to HRP in the presence of hydrogen peroxide. The reaction is stopped by the addition of sulfuric acid (H2SO4), and the absorbance of the resulting yellow product is measured at 450 nm. Absorbance is directly proportional to the concentration of UCHL1 in the samples and standards. A standard curve is generated by plotting absorbance values against concentrations of the standard, and concentrations of unknown samples are calculated using the standard curve.

Reference: Doran JF, Jackson P, Kynoch PA, Thompson RJ. Isolation of PGP 9.5, a new human neurone-specific protein detected by highresolution two-dimensional electrophoresis. J Neurochem. 40:1542-7 (1983).