While Western blotting is a relatively simple and straightforward technique, it does not always yield results that meet with expectations . When this occurs, it is useful to be able to quickly isolate the possible causes and to formulate an effective solution by troubleshooting the experiment . In this chapter, suggestions are offered to assist in identifying and resolving some frequently encountered problems that arise during the course of Western blotting.
Many of these problems can be avoided with careful attention to experimental protocols, and with optimization at key stages throughout the procedure . As mentioned at the end of Chapter 3, the proper antibody concentration can be determined with the use of dot blots and slot blots, significantly reducing the need for future troubleshooting .
Additionally, the use of negative and positive controls are of great help in assessing where things have gone wrong, and eliminating some possible sources of error under consideration . Since one issue that commonly arises during Western blotting is the presence of unusual or unexpected bands on the blot, our troubleshooting section begins with a table describing some common reasons and potential solutions for addressing and evaluating this type of problem .
In most cases, the rest of the troubleshooting issues can be grouped into three major types: no bands, faint bands, and signal on Western blots that interferes with bands . Therefore, the potential causes and solutions have been organized in this manner, and also with regard to potential problem sources, i.e. antibody, antigen, technique, or buffer related where applicable .
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|Chapter 5: Western Blot Protocol: Immunodetection - Direct and Indirect||Western Blot Troubleshoot: Unusual or Unexpected Bands|