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Transfer and multiplex immunoblotting of a paraffin embedded tissue
Author(s) -
Chung JoonYong,
Braunschweig Till,
Baibakov Galina,
Galperin Mike,
Ramesh Arun,
Skacel Marek,
Gannot Gallya,
Knezevic Vladimir,
Hewitt Stephen M.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401343
Subject(s) - multiplex , tissue microarray , antibody , protein microarray , antibody microarray , epitope , proteome , immunofluorescence , microbiology and biotechnology , biology , primary and secondary antibodies , immunohistochemistry , computational biology , chemistry , microarray , gene expression , bioinformatics , biochemistry , gene , immunology
As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho‐specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.