Transfer and Multiplex Immunoblotting of a Paraffin Embedded Tissue

Proteomics. 2006 Feb;6(3):767-74. doi: 10.1002/pmic.200401343.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Antibodies, Phospho-Specific
  • Epithelium / metabolism
  • Gene Expression Profiling
  • Humans
  • Immunoblotting / methods*
  • Immunoenzyme Techniques
  • Keratins / metabolism
  • Microarray Analysis
  • Neoplasm Invasiveness
  • Neoplasm Proteins / analysis*
  • Paraffin Embedding*
  • Phosphoproteins / analysis
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, Retinoic Acid / immunology
  • Receptors, Retinoic Acid / metabolism

Substances

  • Antibodies, Phospho-Specific
  • Neoplasm Proteins
  • Phosphoproteins
  • Receptors, Retinoic Acid
  • retinoic acid receptor beta
  • Keratins
  • Proto-Oncogene Proteins c-akt