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Methods Enzymol. 2009;463:573-99. doi: 10.1016/S0076-6879(09)63033-0.

Performing and optimizing Western blots with an emphasis on chemiluminescent detection.

Author information

1
Thermo Fisher Scientific, Pierce Protein Research, Rockford, Illinois, USA.

Abstract

Immunodetection refers to any detection method that exploits the interaction of an antibody and antigen. The choice of detection method, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, depends on the researcher's preferences and requirements. If a researcher wants to quantify a low-abundance target protein then a chemiluminescent ELISA is used. If a researcher wants to identify a protein that is in high abundance, a colorimetric Western blot will suffice. If there are multiple targets within an assay, then multiplex fluorescence is typically used. This article focuses on Western blotting. Although colorimetric and fluorescent detection methods are discussed, chemiluminescent detection is used most often and is, therefore, discussed in great detail. Included is specific information about the chemiluminescent signal and factors that affect its intensity and longevity. We also describe types of blotting and present data and suggestions for obtaining semiquantitative data. Although classical Western blotting is typically used for qualitative purposes, we present information about effective quantitative analysis using specific controls. Common occurrences within the methodology and their possible explanations are also detailed. One frequent result is the appearance of ghost bands, which, based on our research, can be caused by high amounts of target or antibody cross-reactivity. Also included are the basic Western blot protocol and protocols for troubleshooting common problems and optimizing many of the specific factors that influence results.

PMID:
19892193
DOI:
10.1016/S0076-6879(09)63033-0
[Indexed for MEDLINE]

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