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Total Protein Normalization: Which stain is the best for you?

Housekeeping proteins: The tried and true target for normalizing Western blots…or is it?

Housekeeping proteins: The tried and true target for normalizing Western blots…or is it?

Recently, Total Protein Normalization (TPN) has been emerging as the new preferred normalization method for quantitative western blots. So if TPN is the new standard, then what are our options? Here we review some commonly known total protein stains and a few that may not be so common.

Staining for Total Protein Normalization

Introduction

Normalizing protein levels during quantitative western blotting controls for a variety of data-skewing scenarios: non-uniform sample preparation, uneven loading, unequal transfer, unintended differences in cell culture conditions and others. Traditionally, researchers have relied on the relative abundance of their protein of interest to that of a housekeeping protein (e.g. GAPDH, β-actin), which serves as a loading or normalization control to ensure that resulting assay data actually represent true biological variation and not experimental artifact. The ratio of the concentration a protein of interest to that of a housekeeping protein assumes that the changes in housekeeping protein concentration are independent of experimental conditions. The flaw in this logic is that the concentration of housekeeping proteins do not always accurately reflect these differences and are, in fact, influenced by a variety of factors.

Many journals now caution against using housekeeping proteins for normalization and require additional validation when used. For example, Associate Editors from The Journal of Biological Chemistry have been quoted promoting the use of total proteins over housekeeping proteins for normalization1:

  • "It is typically better to normalize Western blots using total protein loading as the denominator"
  • "We prefer that signal intensities are normalized to total protein by staining membranes with Coomassie Blue, Ponceau S, or other protein stains, and we strongly caution against the use of housekeeping proteins for normalization, unless there is a clear demonstration that expression of the housekeeping protein is unaffected by the experimental conditions."

This is further highlighted in the JBC "Instructions for Authors"2.

Total protein normalization is emerging as the preferred method for quantitative western blot normalization, but what exactly is TPN? TPN quantifies the abundance of the target protein to that of all the protein in that specific lane. This way, normalization doesn’t rely on the potential variation in a single housekeeping protein, but rather the concentration of all proteins within that lane. This addresses many of the disadvantages of relying on individual housekeeping proteins.

There are a number of ways to perform TPN and all are based on staining all the proteins within a gel so they can be visualized. The most common are arguably Coomasie Blue and Ponceau S. But are these our only options? Are they all the same? Which stain should we use? The short answer: It depends. You should use the stain that is best for your specific experimental parameters. Let us explain:

TPN and total protein staining

Total protein stains are designed to bind all proteins, essentially labeling them for visualization. There are several stains available including dyes (e.g. Ponceau S and Coomassie Brilliant Blue), silver stains, fluorescent dyes (e.g. Sypro Ruby), stain-free technology and QuickStain technology.

Staining Type Sensitivity Reproducibility Process Dedicated System Dynamic Range
Ponceau S ~100ng Low Medium N/A Narrow
Coomasie (classical) ~100ng Low Medium N/A Narrow
Silver ~1ng Low Long N/A Narrow
Sypro Ruby ~1ng Low Short Fluorescent Reader only High
Stain-Free (pre-cast) ~1ng High N/A Dedicated gel system & UV source required Medium to High
QuickStain Sub-ng High Single incubation step Fluorescent Reader only High
  • Ponceau S

    A reversible dye that stains proteins red, is easily removed by washing, easily applied and cost efficient. Ponceua S intensity, however, decreases quickly over time, so detection should be conducted rapidly. This high time sensitivity to staining intensity also results in poor reproducibility.

    • Pros: Cheap, reversible, ideal for some downstream applications such as blot-sequencing
    • Cons: Intensity decreases rapidly, poor reproducibility
  • Coomassie Brilliant Blue (classical)

    A family of common dyes (G-250 and R-250), that binds proteins primarily through arginine, lysine and histidine and stains them bright blue. Coomassie staining is irreversible but ideal for some downstream applications such as Mass Spectrometry of excised bands. However, Coomassie staining reproducibility is hampered by the lack of standardization in the destaining step. Further, Coomassie has is not ideal for low abundant proteins <100ng.

    • Pros: Common, cheap, ideal for some downstream applications such as Mass Spec
    • Cons: De-staining isn’t always uniform, lack of reproducibility, not ideal for low abundant proteins
  • Silver Stain

    Silver stain is ideal for applications in which high sensitivity (<1ng) is needed for low abundance proteins and there is no requirement for downstream applications (due to irreversible formadehyde fixation). Silver ions bind proteins and are then reduced to silver metal via a developing solution, creating a visible image. Silver staining process is complex and contains multiple steps and reagents, making the process time consuming. Reproducibility is also hampered since the time to develop is variable between gels.

    • Pros: Ideal for low abundant proteins (<1ng)
    • Cons: irreversible binding, bad for downstream applications, long and complex process, poor reproducibility, not ideal for quantitation due to narrow dynamic range, toxic chemicals required
  • Sypro Ruby

    Sypro Ruby is a fluorescent dye that bindings selectively to basic amino acids (lysine, arginine and histidine). Sypro Ruby has very high sensitivity that is comparable to Silver Staining, but without the complex protocol. Unlike Silver staining, however, the use of fluorescent dyes requires specialized equipment to visualize proteins.

    • Pros: high sensitivity (<1ng), short process, no impact to downstream applications
    • Cons: Requires fluorescent reader, expensive
  • Stain-Free Technology

    Stain free total protein detection leverages a proprietary Trihalo™ compound to create a unique gel chemistry. Trihalo™ containing gels results in modification of tryptophan and results in a fluorescent signal. Stain-free technology sensitivity is equivalent to that of silver staining, with sensitivity for tryptophan-containing proteins to be greater. Other benefits are the relative high degree of reproducibility and compatibility with most downstream applications that are hampered by other staining methods such as silver staining. Areas of concern, however, include pre-cast, stain-free gels that can many times require a dedicated system and the inability for this technology to bind proteins with minimal or no tryptophan residues (e.g. aprotinin).

    • Pros: Easy to use pre-cast gels, highly reproducible, does not impact downstream applications
    • Cons: Expensive option, typically requires dedicated gel system, requires proteins that contain Tryptophan, limited sensitivity, requires UV source
  • QuickStain Technology

    QuickStain provides very high sensitivity (sub-ng levels) with the ease of use of stain-free technology but without the stain-free technology limitations. QuickStain relies on a proprietary compound that binds to all proteins in a sample during a 5 minute incubation prior to gel electrophoresis. Stain-free technology sensitivity is partially dependent on the concentration of tryptophan within the protein. Further, there is no required fixation, staining or destaining steps that can inhibit potential downstream applications.

    • Pros: Very high sensitivity (sub-ng), easy to use platform (pre-mix sample before loading), reproducible, does not impact downstream applications, works with any gel system
    • Cons: Requires 5 min incubation prior to electrophoresis, requires fluorescent reader

References:

  1. Foshang, A.J. (2015) Transparency is the Key to Quality Journal of Biological Chemistry 290, 29692-29694
  2. Immunoblots (Western blotting) - http://www.jbc.org/site/misc/ifora.xhtml#blots

Additional Resources