Methods and considerations for quantitative Western blotting using SuperSignal ® Chemiluminescent Substrates

Classical Western blotting is considered a qualitative method; however, it can be used for quantitative analysis of proteins pro-vided that specific controls are included. Using SuperSignal ® West Dura Chemiluminescent Substrate we qualitatively and quan-titatively detected changes in the regulation of IkBα and p53 in A431 cells treated with EGF (and doxorubicin or cisplatin) by comparing to a standard curve generated using pure protein. ELISAs for both proteins enabled confirmation of the Western data. Additionally, using an imager provided greater linear range than film and, therefore, greater flexibility. SuperSignal West Sub-strates are therefore useful for qualitative and quantitative Western blotting when the proper controls are used