Common mistakes in Western blot

High specificity and sensitivity are the key enabling characteristics that made Western blot the gold standard approach for protein detection and quantification. However, poor understanding of the physical and chemical basis of the technique results in common mistakes that could seriously affect the reliability of blot readout. Specifically, issues concerning poor gel casting could result in non-homogeneous distribution of pores between gel lanes, that in allowing faster migration of proteins in some lanes compared to others, result in a wavy line of detected bands. This could impact on the identification of the target protein through the molecular weight marker. Secondly, although overloading of samples may not seriously impact on protein detection, it generates blots which could not be used for the quantification of relative abundance of proteins. Another problem is the non-specific binding of non-target proteins by primary antibodies which manifest as multiple bands on the same membrane strip. This came about due to the presence of multiple binding clefts on the antibody, which could bind to different proteins. Finally, incomplete washing of secondary antibody yields blots with large smear and dark patches that makes the quantification of relative abundances of proteins in different samples difficult. Hence, although Western blot is a standard technique in the life sciences for the specific detection and semi-quantitative analysis of protein abundance, multiple issues ranging from gel casting to sample loading and choice of antibody could confound blot readout. However, simple steps exist to ameliorate the aforementioned problems. For example, greater care in gel casting would enable proteins to migrate uniformly across different gel lanes. Use of the Coomassie Blue stain would provide an indication of the relative abundance of different proteins separated by electrophoresis and help select a suitable dilution factor for a full Western blot. Greater care in washing and use of an appropriate secondary antibody concentration would reduce high background on the membrane strip. But, the most difficult issue remains the choice of primary antibody as it is hard to predict possible non-specific binding partners of the antibody. Overall, Western blot remains a reliable method for the confirmation of target proteins probed by mass spectrometry methods.