Transfer and multiplex immunoblotting of a paraffin embedded tissue.
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.
Chung
Joon-Yong
JY
Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Braunschweig
Till
T
Baibakov
Galina
G
Galperin
Mike
M
Ramesh
Arun
A
Skacel
Marek
M
Gannot
Gallya
G
Knezevic
Vladimir
V
Hewitt
Stephen M
SM
D045163:Antibodies, Phospho-Specific; D004848:Epithelium / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000378:metabolism; D046228:Microarray Analysis; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000032:analysis*; D016612:Paraffin Embedding; D010750:Phosphoproteins / Q000032:analysis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000276:immunology / Q000378:metabolism
D045163:Antibodies, Phospho-Specific; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D007633:Keratins; D051057:Proto-Oncogene Proteins c-akt