As with all techniques, western blotting has its limitations [4,5,11]. The main limitation of western blotting is that it can only be carried out if a primary antibody against the protein of interest is available. To detect post-translational modifications such as phosphorylation of target proteins, specific antibodies against the phosphorylated residues are needed. While antibodies for many different proteins are available from biotech companies, they are not cheap, and if primary antibodies are not available for a given protein, it will not be possible to perform a western blot to detect that particular protein. Another major limitation is that many antibodies exhibit off-target effects by interacting with other proteins. There are also many commercially available antibodies that do not detect the target protein when tested in the laboratory with particular tissues or cell types, resulting in what can only be described as expensive buffer. Another important limitation of the western blotting technique is the technical demand on the scientist. It is not uncommon for simple mistakes such as using too little or too much primary antibody to result in unusable results. An investigation of quantitative western blotting using erythropoietin showed that the interoperator variability was the main error source accounting for nearly 80% of the total variance [12]. Other western blotting limitations include the need for each antibody to be independently optimized and the cost of modern western blotting equipment such as advanced digital imagers. The basic western blot protocol is often ineffective in detecting a particular protein and modified protocols exist in most laboratories. One problem that may be encountered is variations in transfer efficiency. Small proteins (< 10kDa) may not be retained by the membrane, large proteins (> 140kDa) may not being transferred to the membrane, and varying gel concentrations may affect transfer efficiency [13]. Other problems include the primary antibody not recognizing the immobilized antigen in its denatured state, the detection signal decaying too quickly and high background to name a few. However, many of these limitations can be overcome with proper experimental techniques.
