Signaling Proteins as Internal Loading Controls

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Besides housekeeping proteins and total protein controls, signaling proteins are another option for normalization. This approach is particularly useful for relative analysis of post-translational modifications such as phosphorylation. The method combines two primary antibodies raised in different hosts: a phospho-specific antibody (or other modification-specific antibody) and a pan-specific antibody that recognizes the target protein regardless of its modification state. Fluorescently-labeled secondary antibodies are used to simultaneously detect and discriminate the two signals with digital imaging. Phospho-signal is then normalized against the total level of target protein, using the target protein as its own internal control.

This is a great strategy to use if you’re studying protein modifications. Bakkenist et al. examined the possibility of binding interference from combined phospho-specific and pan antibodies, but detected little or no effect.
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Advantages of Phospho-Analysis with Signaling Proteins:

  • You can detect both unmodified and modified forms of your target protein on the same blot, in the same lane.
  • No error is introduced by stripping and reprobing. Stripping and reprobing of blots can introduce detection artifacts and cause loss of blotted proteins from the membrane.
  • Accuracy is improved by correcting for loading and sampling error

Find out more about multiplex analysis using signaling proteins: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Saturation Limits Accurate Western Blot Normalization

Normalization Webinar InvitationFor more information on Western blot normalization, watch these webinars:


An effective loading control has a linear, proportional response, meaning the signal intensity of the internal control should accurately reflect sample concentration and abundance of loading control over a wide range. If your loading control doesn’t meet the requirement of a linear response, it affects your accuracy and reproducibility.

Saturation limits the accuracy of normalization, especially if you’re using a housekeeping protein. Housekeeping proteins are often highly abundant in samples, which can lead to strong, saturated signals.

Let’s look at what saturation is and where it can happen.

What is Saturation?

Saturation is when strong signals don’t accurately reflect protein levels. It can come from your membrane, your detection chemistry, and the way you image your blot.

Saturated bands are deceptive (Fig. 1). They hide actual variation in protein levels and underestimate the amount of protein present. The similar apparent intensities of saturated bands may lead you to think your protein levels are equal.
Blog Post 4 - Normalization
Figure 1. Strong bands become saturated and underestimate protein abundance. Strong signals (box) exhibit saturation because they fall outside the linear range of detection. Band intensity can no longer increase proportionately to indicate protein abundance. As a result, the signal intensity of the saturated bands appears similar. High-intensity data points should not be used as controls for normalization.

Membrane Saturation

If you’ve overloaded the samples on your gel, that problem doesn’t go away once you transfer to the membrane. You may lose protein while transferring to the membrane, if overloaded samples exceed membrane capacity.

In addition, highly abundant proteins might stack on top of each other. When primary antibodies can only access the top layer of the protein stack, they can’t detect the rest of the proteins. This leads to underestimation of strong signals, hurting accurate quantitation.

How can you prevent membrane overloading? It’s best to run a dilution series to determine the upper limit of how much sample you should be loading on your gel. Membrane overloading is tricky to avoid, because different proteins generally have different upper limits in the same sample. Because it arises from the binding chemistry of proteins and blotting membranes, membrane saturation can happen with any detection chemistry or imaging method.

Detection Chemistry Saturation

When internal loading control bands are detected outside the linear range of detection, increases in protein level won’t produce a proportional increase in signal intensity. For accurate normalization, both the internal loading control and the target must be detected within the linear range of the method used. The type of detection chemistry you use affects the linear range of detection for your sample proteins.

Enhanced chemiluminescence (ECL) is an indirect, enzymatic method. Secondary antibodies are labeled with horseradish peroxidase (HRP) as an enzymatic reporter. The enzyme produces light after you apply substrate and produces an unstable, time-dependent signal. Because these signals are the result of the kinetics of an enzymatic reaction, the signal doesn’t reflect its protein abundance. Saturation is likely with ECL, because it amplifies signals.

Fluorescence, on the other hand, is direct detection. Fluorophores label secondary antibodies and then generate stable signals. This type of detection chemistry doesn’t depend on enzyme kinetics, so fluorescent detection is more reproducible than ECL detection. Fluorescence is also less likely to saturate, because the signals are directly proportional to the amount of protein.

How can you prevent detection chemistry saturation? The simplest way is to use fluorescence detection instead of ECL, because fluorescence is less likely to saturate.

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