Abstract
Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.
Highlights
Mapping of the human proteome in normal and diseased cells will greatly increase our understanding of many aspects of cell biology, for example, differentiation and disease development, because proteins constitute the functional elements in most cell biological processes
There are mainly two different strategies used for mapping of the human proteome: One is separation-based proteomics, which uses electrophoresis or liquid chromatography in combination with mass spectrometry to study proteins in complex bio samples, whereas the other is based on usage of affinity proteins to study proteins in various applications, for example, western blotting (WB) and immunohistochemistry (IHC)
We found that antibodies biotinylated with the ZBPA technique show a staining pattern in concordance with their unconjugated counterparts
Summary
Mapping of the human proteome in normal and diseased cells will greatly increase our understanding of many aspects of cell biology, for example, differentiation and disease development, because proteins constitute the functional elements in most cell biological processes. There are mainly two different strategies used for mapping of the human proteome: One is separation-based proteomics, which uses electrophoresis or liquid chromatography in combination with mass spectrometry to study proteins in complex bio samples, whereas the other is based on usage of affinity proteins to study proteins in various applications, for example, western blotting (WB) and immunohistochemistry (IHC). IHC allows for in situ visualization of protein expression in tissues and is a valuable tool in clinical pathology. The specificity and sensitivity with which an antibody binds its intended target is of fundamental importance to achieve reliable data.
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