Here, we describe some general techniques to produce good quality protein samples with minimal degradation for improved reproducibility between experiments. Alternatively, normalization by total, blot-transferred protein has recently been shown to give excellent data for typical total protein lysates. Also, these HKPs can be affected by the treatment conditions of the experiment giving skewed results for target protein expression that do not reflect the biology of the tested samples. The direct effect of poor normalization is evident when sample loading above 10 μg of a total protein lysate per lane is required because traditional loading HKPs such as GAPDH, actin, and tubulin are grossly overloaded and therefore not serving the purpose of data normalization. Thus, data normalization is crucial to identify and correct experimental errors where reference instability becomes increasingly important with the measurement of smaller differences in target protein expression between samples. Furthermore, the appropriate selection of normalization method (based on reference signals obtained either by housekeeping proteins (HKPs) after immunochemical staining or total protein (TP) intensity on blotting membranes after total protein staining) must be considered to assure that the reported fold changes of the target protein are not an artifact of reference signal. Briefly, the validation of antibodies (Ab) is critical both to assure that the Ab/antigen interaction is specific and correct and to determine the dilution factor of samples that is required for protein loading in the quantitative linear dynamic range for each antibody. In order to obtain quantitative data from western blots, a rigorous methodology must be used as previously described. Although labs have been quick to purchase the latest detection technologies and reagents for western blotting, the associated techniques used to produce the densitometric data have not evolved leading to published data that are difficult or impossible to interpret or reproduce. In addition, the ongoing development of detection media and reagents has provided the scientific community with ultrasensitive imaging systems giving broad dynamic range of detection enabling precise and accurate quantitation of signals from both low and high expressing proteins from the same blot. Over the past three decades, the sensitivity, robustness, and flexibility of the corresponding indicator systems have increased significantly. in 1979 and has since become a common technique used in research laboratories globally for the immunodetection and quantitation of specific proteins in complex cell homogenates. Western blotting was introduced by Towbin et al. The resulting data are typically confirmed by a second, independent method such as western blotting. Proteomic technologies such as two-dimensional electrophoresis and mass spectrometry are valuable tools in semiquantitative protein profiling studies in order to identify broad expression patterns enabling a better understanding of molecular events, signaling pathways and mechanisms. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample.

Loading control western blot