The Importance of Detecting in the Combined Linear Range for Western Blots



In the instructions to authors for the Journal of Biological Chemistry, they state:

What is the linear range of detection?

In quantitative Western blot analysis, the linear range of detection is the range of sample loading that produces a linear relationship between the amount of target on the membrane and the band intensity recorded by the detector.

Within the linear range of detection, band intensity should be proportional to the amount of target. A change in target abundance should produce an equivalent change in signal response. At the upper and lower ends of the linear range, this proportional relationship is lost. Band intensity no longer reflects the abundance of target, and quantification is not possible.

Quantitative Western blot analysis is only accurate if the target protein and internal loading control can both be detected within the same linear range – a range that must be determined experimentally for each target and loading control. The combined linear range is then used to determine how much sample should be loaded to produce a linear signal response for both the target protein and the internal loading control.

Are YOU detecting your target protein and your internal loading control in the combined linear range?

How is the combined linear range determined?

Help has arrived! The protocol “Determining the Linear Range for Quantitative Western Blot Detection” from LI-COR explains how to use serial dilutions of sample protein to determine the linear ranges of detection for a target and internal loading control, and choose an appropriate amount of sample to load for quantitative Western blot analysis.

This protocol also explains key factors for success, required reagents, data analysis and interpretation. Two methods for determining the linear range are included in the protocol:

  • Determining the Linear Range for a Target Protein and REVERT™ Total ProteinStain. Follow these instructions if total protein staining of the membrane will be used as the internal loading control for quantitative Western blot normalization.
  • Determining the Linear Range for a Target Protein and a Housekeeping Protein. Follow these instructions if a housekeeping protein will be used as the internal loading control for quantitative Western blot normalization. This method also applies to normalization with a pan-specific antibody for analysis of phosphorylation or other post-translational modifications.

LI-COR has several other protocols to help you get published. In all of them, key factors for success, data analysis and interpretation are covered as well as links to additional educational resources.

With these protocols and our scientific experts, we can help you collect rock-solid data that will meet even the toughest publication standards. Protocols are also available in an online format at protocols.io

Download your copy of Determining the Linear Range for Quantitative Western Blot Detection so that you can accurately determine the linear range for your quantitative western blot detection. Let us help you be confident in the Western blotting data you submit for publication.