The Gold Standard for Western Blot Normalization: Total Protein Staining

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

While you have choices for your Western blot normalization strategy – you can still use housekeeping proteins as long you have validated that their expression is not changing – total protein staining detection is becoming the “gold standard” for normalization of protein loading.

After transfer, but prior to immunodetection, the membrane is treated with a total protein stain to assess actual sample loading across the blot. Because this internal loading control uses the combined signal from many different sample proteins in each lane, error and variability are minimized. This antibody-independent method corrects for variation in both sample protein loading and transfer efficiency, and monitors protein transfer across the blot at all molecular weights. The figure at the left shows that REVERT Total Protein Stain provides highly efficient protein staining on nitrocellulose or Immobilon®-FL PVDF membranes in under 10 minutes. Complete figure legend.

REVERT™ Total Protein Stain is a near-infrared fluorescent membrane stain used for total protein detection and normalization. REVERT staining is imaged at 700 nm, and fluorescent signals are proportional to sample loading.

The REVERT Total Protein Stain Normalization protocol describes how to use REVERT Total Protein Stain for Western blot normalization and quantitative analysis. It includes step-by-step instructions on how to use REVERT stain. There is also detailed information on normalization calculations, analysis of replicates, and data interpretation.

Replication is an important part of quantitative Western blot analysis and is used to confirm the validity of observed changes in protein levels. Biological and technical replications should both be done, since they are both important but meet different needs.

LI-COR has several other protocols to help you meet publication guidelines and requirements. 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 accurate, reliable data that will meet even the toughest publication standards. Protocols are also available in an online format at

Download your copy of REVERT Total Protein Stain Normalization protocol and use the gold standard to determine your protein loading concentrations. Let us help you be confident in the Western blotting data you submit for publication.

New Protocols for Western Blot Normalization to Help You Get Published

Western blotting is the most widely used method for the detection and characterization of proteins. Although the basic elements of Western blotting remain unchanged, journal standards for publishing Western blots (e.g., JBC’s Instructions for Authors) have become more rigorous in recent years.

Are you interested in quantifying your proteins on your Western blot but are not sure how to manage Western blot variability and increase the accuracy of your results?

The key is to maximize Western blot accuracy and precision. This makes relative comparisons meaningful. How can you accomplish this? By reducing variability whenever possible with good experimental design. You can also correct for variability by using the appropriate internal loading controls for your Western blot normalization.

Normalization Protocols

LI-COR developed a series of protocols to help improve the quality of quantitative Western blots. Whether you are a beginner or a seasoned user, we can help you collect rock-solid data that will meet even the toughest publication standards.

The protocols cover key factors for success, data analysis and interpretation, and include links to additional educational resources for quantitative Western blotting.

Do you need help determining the linear range of your target protein and internal loading control, or validating your housekeeping protein, using REVERT total protein stain for normalization or using total and post-translationally modified proteins for normalization? If so, our tools, products, and services can help you get published.

These protocols are also available in an online format at

Total Protein Stain as an Internal Loading Control

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

Using a total protein stain to detect the total protein in each lane of your gel or blot is becoming more popular. Total protein staining is a direct measure of the total amount of sample protein in each lane. For each lane, the sum of all the signal intensities of all the proteins in the lane is used for normalization.

This more direct approach may increase the accuracy of normalization. Unlike housekeeping proteins, total protein staining does not require validation for each experimental context.

A total protein stain should produce a linear increase in signal intensity in response to increasing protein concentration. It should also correct for variation at all points in the Western blot process, including gel loading and transfer to membrane. It must be compatible with downstream immunodetection of your blot. You should make sure that the signal intensity of the total protein stain is moderate, without saturation or low signal-to-noise ratios.

REVERT™ Total Protein Stain provides linear, proportional signal across a broad range of sample concentrations.

REVERT Total Protein Stain

Learn more about total protein controls in the full paper on normalization: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Signaling Proteins as Internal Loading Controls

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

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.
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.
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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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Understanding Western Blot Normalization

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

chess piece - kingResearchers rely on Western blotting to detect target proteins in complex samples. This trusted technique is widely used to compare relative protein levels.

However, variability can creep into your Western blots through differences in sample preparation, sample loading, and transfer from gel to membrane. That’s why normalization is important. Normalization is the process of using internal loading controls to mathematically correct for sample-to-sample variation. These internal loading controls verify whether or not samples are uniformly loaded across the gel, confirm consistent transfer from gel to membrane, and enable comparison of relative protein levels between samples.

Normalization is meant to correct for small variation between samples, and can’t completely remove variability. If large data corrections are applied, accuracy may be affected. Normalization is a strategy to apply throughout your experiment, rather than a last step in the protocol. The more sources of variability you can reduce or eliminate, the more reproducible your experiment will be.

The role of an internal loading control is always to confirm the changes you see on the blot reflect actual change in the biology of your samples. To demonstrate statistically significant changes in the abundance of target protein, you need a reliable normalization strategy that fits the context and biology of your experiment. Effective, carefully-planned normalization will more accurately reflect the amount of protein in each lane.

chess piece - bishopUnderstanding Western blot normalization will help you choose a strategy that fits the context and biology of your experiment.

This paper describes important considerations, strengths, and limits of commonly used normalization strategies:

Western Blot Normalization: Challenges and Considerations for Quantitative Analysis