Do you have questions about Western blot normalization? Get practical, expert advice on how to normalize from Steve Shiflett, LI-COR product manager.
An effective loading control will display a linear relationship between signal intensity and sample concentration. Saturation can often prevent this linear response, especially for highly abundant proteins. A quick recap: saturation is when strong band intensities appear different, but relative signal intensity plateaus. Check out a previous blog post on how saturation limits accurate Western blot normalization.
Linear range is the region over which signals are directly proportional to the amount of protein present. A wider dynamic range makes it easier to get data within the linear range today, as well as next year – increasing reproducibility.
Film Exposure of Chemiluminescent Blots
While film might be the method of choice for some researchers, it has fundamental limitations that affect the analysis and reproducibility of your data. It provides an extremely narrow linear range of detection, roughly 4-10 fold. Also, rapid saturation of strong signals makes it difficult to accurately determine the upper limit of detection. Film exaggerates small differences in abundance and masks sample-to-sample changes in strong bands.
Figure 1. Odyssey® data are linear across a much wider range than ECL and film. Pure recombinant p53, Hdm2, and Hdmx protein of known concentration were serially diluted and run in duplicate, followed by Western blot analysis. Proteins were detected by IR fluorescence or standard ECL. Signal intensities were quantified with Odyssey software or, for ECL, densitometry of developed films. Reprinted from Wang, YV et al. Proc Natl Acad Sci USA. 104(30): 12365-70 (2007). Copyright (2007) National Academy of Sciences, U.S.A.
CCD Imaging of Chemiluminescent Blots
Digital imaging of chemiluminescent blots typically offers a wider linear range of detection than film. Many CCD systems are able to detect faint signals without saturating strong signals. Sensitivity and linear range depend on which CCD system you choose.
Even with a digital imager, chemiluminescent Western blot signals are still the result of an enzymatic reaction. The time-dependent enzymatic reaction may still lead to saturation and inaccurate results.
Digital Imaging of Fluorescent Blots
Fluorescent immunoblotting is best performed with near-infrared fluorescent dyes and imaging systems. Background autofluorescence of membranes and biological samples is low in the near-infrared region, enabling high sensitivity. To detect faint signals without saturating strong signals, use an imaging system with a wide linear dynamic range.
Are you experiencing detection system saturation? Find more information about saturation in this full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis