Are genuine changes in protein expression being overlooked? Reassessing Western blotting

This study used purified calsequestrin I and AMP kinase (AMPK) proteins to demonstrate how Western blotting outcomes can be influenced when either the density of proteins detected lie within a nonproportional region of a standard curve or a standard curve is not taken into account for data analyses. It outlines the likelihood of true changes being overlooked through the simple mistake of using band density alone and/or through analyzing too much sample. To demonstrate this, extrapolation of a typical linear, although non proportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta 1 in rat extensor digitorum longus muscle as being of the order of 60 mu M. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.