摘要：

Peptidyl-prolyl cis-trans isomerase (Pin1), is also known as PPIase, PPIase Pin1, Roamase PIN1, and Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, is a 163 amino acid nuclear protein with a calculated molecular weight of 18.2 kDa. Pin1 has been implicated in neurodegerative disorders and is over-expressed in many human cancers. Pin1 has been described as a catalyst for oncogenesis and called a potential therapeutic target in cancer. Pin1 is involved in many basic cellular processes including cell-cycle regulation, transcription, differentiation and proliferation. Pin1 belongs to the parvulin family within the peptidyl-prolyl cis-trans isomerase (PPIase) group of proteins. Pin1 consists of a C-terminal PPIase domain and an N-terminal WW domain. The WW domain binds a specific motif of phosphorylated serine or threonine residues preceding a proline and catalyses the cis-trans isomerisation of the proline-containing protein. This report details the development of a sandwich ELISA for the quantification of human Pin1 from cell lysates. The assay uses recombinant human Pin1 to generate a standard curve. A capture antibody is immobilized to 96 well microtiter plates and stored at 4 degrees. A rabbit anti-human Pin1 polyclonal antibody is used as a detection antibody. A goat anti-rabbit horseradish peroxidase conjugate follows and then tetramethylbenzidine (TMB) is utilized as a substrate. Color development is stopped by the addition of 1N HCl. The assay requires 3 hours to complete. Cells lysed in RIPA buffer require a 1:4 dilution in assay buffer prior to application in the immunoassay. The assay has a standard curve range of 8000 pg/mL to 250 pg/mL. The assay shows no cross reactivity with human PCNA, BAD, beta-catenin or BCL-2. Immunoassay results parallel western blot data. The immunoassay may become a valuable tool for the study of human Pin1 levels associated with various disease processes including neurodegnerative diseases and cancer.

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