The design of a quantitative western blot experiment

Abstract

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. 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. 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. 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. 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.

Figure 1

Reliable western blot data can only be generated when the proper sample amount of protein is used. Loading too much protein leads to signal saturation in western blots, yet too little produces weak signals. Once the experimental setup and conditions are established for the assay, do not change the sample load, transfer method, transfer time, antibody dilution, antibody incubation time, or temperature in subsequent experiments as these factors may significantly change the detection signals.

Figure 2

(From [9] with permission from the authors and Bio-Rad.) Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10–50 μg of HeLa cell lysate. On the left are representative images of (a), stain-free blot and the chemi blots for (b), β-actin; (c), β-tubulin and (d), GAPDH. Lane labels correspond to total protein load (μg). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted “quantitative response” of actual loading whereas the stain-free signal correlated to the expected result (e).

Figure 3

(From [8] with permission from the authors and Bio-Rad.) Image acquisition and densitometric analysis. Image Lab software version 5.0 (Bio-Rad) was used for image acquisition and densitometric analysis of the gels, blots, and film in this study. The software interprets the raw data in three dimensions with the length and width of the band defined by the “Lanes and Bands” tool in concert with the “Lane Profile” tool such that the chemiluminescent signal emitted from the blot is registered in the third dimension as a peak rising out of the blot surface. The density of a given band was measured as the total volume under the three-dimensional peak, which could be viewed in two dimensions using the “Lane Profile” tool to adjust the precise width of the band to account for the area under the shaded peak of interest. Background subtraction was set by using the rolling disc setting in the “Lanes” tool. The rolling disc values were set such that the background was subtracted under the band (i.e., peak) of interest in a uniform manner between the lanes of a given blot. In this case, the rolling disc for the two lanes analyzed was set to 18 and 25, respectively, such that the peaks of interest were cut at a consistent level between the markers shown with an “X”.