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Normalization with a
Total Protein Control

What is a Total Protein Control?

With a total protein control, you correct for sample-to-sample variation using all proteins present in the sample. There are two types of total protein controls – irreversible covalent labeling and total protein staining. Understanding the limitations of each Western blotting normalization strategy will help you select the best method for accurate analysis. The key thing to remember is: are you reducing error and variability, or introducing it with your chosen strategy?

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Irreversible Covalent Labeling

Stain-Free™ and CyDye methods fall into the category of irreversible covalent labeling. Both of these methods chemically alter the protein samples, which may interfere with immunodetection.


This method uses a commercial pre-cast gel that contains trihalo compounds. UV light causes these compounds to irreversibly cross-link with tryptophan residues present in your sample. Limitations of this method include low sensitivity for membrane detection, chemical modification of your sample, and a less flexible workflow than other methods.

stain-free detection

Taylor SC et al. 2013. ​Mol Biotechnol. 55:217-26.

stain-free protein figure

All sample proteins

Target protein

Trihalo compounds in pre-cast gel

ECL detection of target

Tryptophan residues


An amine-reactive fluorescent dye pre-labels the total protein samples by covalently binding to lysine residues within your sample. Limitations of this method include possible interference with antibody binding during immunodetection, a nonlinear response to labeling, and irreversible chemical modification.

cydye protein figure

All sample proteins

Target protein

Fluorescent detection of target protein

Covalent labeling with fluorescent dye

Lysine residues

Total Protein Staining

This approach measures the total protein in each lane. Ideally, total protein stains will have a linear signal output in response to sample concentration, correct for variation at all points in the Western blotting process, and be compatible with immunodetection of target proteins.

“Normalization of signal intensity to total protein loading (assessed by staining membranes using Coomassie blue, Ponceau S, or other protein stains) is preferred.”

“Instructions for Authors.” The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 3 March 2016.

REVERT™ Total Protein Stain

REVERT Total Protein Stain is a membrane-based (post-transfer) normalization strategy that stains all protein in your sample. No special reagents, equipment, or gels are required with REVERT Total Protein Stain, so it is compatible with your Western blot protocol.

REVERT staining does not interfere with immunodetection, and can be easily reversed under alkaline conditions. Other total protein controls like Stain-Free and CyDye chemically modify your target proteins. These irreversible changes can interfere with downstream immunodetection (by blocking antibody binding).

Read More about REVERT Total Protein Stain

REVERT protein figure

All sample proteins

Target protein

Reversible total protein stain

Fluorescent detection of target

700nm and 800nm Western Blot

Normalize for Your Needs

When versatility is what you need, go with the normalization strategy that is best for you and your research. Regardless of your experimental conditions, Odyssey® imaging systems can provide accurate results based on any normalization strategy.

More about the Odyssey CLx Imaging System

“The brilliant signal-to-noise ratio in combination with the ability to truly quantify the data is really outstanding.”

Geir Bjørkøy, University College of Sør-Trøndelag

Internal Reference Protein

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