The Gold Standard for Western Blot Normalization: Total Protein Staining

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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 protocols.io

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.

Do you have a question about your Western blot normalization strategy, the importance of replicates. or how publication requirements for Western blots have been changing? Contact us and let our experts help!

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

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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.

Do you have a question about your Western blot normalization strategy or how publication requirements for Western blots have been changing? Contact us and let our experts help!

New Protocols for Western Blot Normalization to Help You Get Published

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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 protocols.io

Do you have a question about your Western blot normalization strategy or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Normalization Is Critical for Quantitative Immunoblotting

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

webinar-2-for-facebookInternal loading controls and normalization are critical for quantitative immunoblotting. An accurate loading control will display a linear relationship between signal intensity and sample concentration. An effective normalization strategy should correct for variability at all stages of the immunoblotting process, including the transfer of sample proteins to membrane. It should also be compatible with immunodetection of target proteins.

As researchers detect and interpret subtle changes in protein samples, accurate normalization is becoming increasingly important.

Odyssey CLx Infrared Imaging SystemThe best normalization strategy is one that fits the context and biology of your experiment. No matter what normalization strategy you choose, an Odyssey® Imaging System can provide quantitative results.

Learn more from the full paper on normalization: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Ready to find out if the Odyssey CLx Imaging System is right for your lab? Request a demo.

Do you have a question about your Western blot normalization strategy or how publication requirements have been changing? Contact us and let our experts help!

Total Protein Stain as an Internal Loading Control

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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

Do you have a question about your Western blot normalization strategy or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Signaling Proteins as Internal Loading Controls

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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.
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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

Do you have a question about your Western blot normalization strategy, the importance of replicates. or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Housekeeping Proteins as Internal Loading Controls

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

Housekeeping proteins such as tubulin, actin, and GAPDH are often used to normalize. In the past, researchers assumed that these proteins were constant in every cell type, because these proteins maintain basic cellular function. Housekeeping proteins are acceptable loading controls if expression is stable, but expression of these proteins can vary depending on your cellular context.

Housekeeping proteins won’t effectively normalize in every experiment, but that doesn’t mean they won’t work for any experiment. If you choose to use a housekeeping protein as your normalization strategy, be sure to validate it to confirm stable expression for your experimental context. As cell types, tissue types, disease states, and experimental treatments change, your internal loading control should remain constant.

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Here are some things to keep in mind:

  • Gene expression levels do not reliably predict protein abundance. Just because mRNA levels are constant, this does not mean protein levels will be similarly constant.
  • Biological factors, like tissue type, growth conditions, stage of development, and disease, may influence expression of housekeeping proteins. Without constant expression, housekeeping proteins are an unreliable way to normalize.
  • Housekeeping proteins are typically very abundant. The resulting strong bands frequently cause signal saturation, which reduces the accuracy of detection.

If you have validated that your housekeeping proteins are constant across all your experimental treatments, you can use them as a reliable loading control. Actin, tubulin, and COX IV primary antibodies can be used for two-color normalization.

Find out more about housekeeping proteins as internal loading controls in Western Blot Normalization: Challenges and Considerations for Quantitative Analysis.

Do you have a question about your Western blot normalization strategy, the importance of replicates. or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Types of Normalization Strategies

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Western Story - GlovedScientistsWhat normalization strategies are available to you when performing Western blots? The next four blog posts in this series will discuss the options that exist and considerations for use of each strategy.

An ideal internal loading control would have a linear, proportional response, be stably expressed in all experimental conditions, correct for variation throughout the whole process of immunoblotting, and be compatible with detection of your target proteins.

If you have the time to validate your loading control for each experiment, a housekeeping protein may work for you. You must validate all your housekeeping proteins to ensure stable expression.

If you’re studying protein modifications, like phosphorylation, ubiquitination, or glycosylation, then a multiplex normalization strategy with a signaling protein is recommended.

Total protein controls use all proteins present in the sample, and include total protein stains. If you don’t have time to validate, a total protein stain is best.

There are many ways to normalize. The best way depends on you and your experimental context. Watch for future blog posts about housekeeping proteins, signaling proteins, and total protein stains.

Find out more about normalization: Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Do you have a question about your Western blot normalization strategy or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Are You Experiencing Detection System Saturation?

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

An effective loading control will display a linear relationship between signal intensity and sample concentration. Saturation can often prevent this linear response, especially for highly abundant proteins. A quick recap: saturation is when strong band intensities appear different, but relative signal intensity plateaus. Check out a previous blog post on how saturation limits accurate Western blot normalization.

Linear range is the region over which signals are directly proportional to the amount of protein present. A wider dynamic range makes it easier to get data within the linear range today, as well as next year – increasing reproducibility.

Film Exposure of Chemiluminescent Blots

While film might be the method of choice for some researchers, it has fundamental limitations that affect the analysis and reproducibility of your data. It provides an extremely narrow linear range of detection, roughly 4-10 fold. Also, rapid saturation of strong signals makes it difficult to accurately determine the upper limit of detection. Film exaggerates small differences in abundance and masks sample-to-sample changes in strong bands.

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Figure 1. Odyssey® data are linear across a much wider range than ECL and film. Pure recombinant p53, Hdm2, and Hdmx protein of known concentration were serially diluted and run in duplicate, followed by Western blot analysis. Proteins were detected by IR fluorescence or standard ECL. Signal intensities were quantified with Odyssey software or, for ECL, densitometry of developed films. Reprinted from Wang, YV et al. Proc Natl Acad Sci USA. 104(30): 12365-70 (2007). Copyright (2007) National Academy of Sciences, U.S.A.

CCD Imaging of Chemiluminescent Blots

Digital imaging of chemiluminescent blots typically offers a wider linear range of detection than film. Many CCD systems are able to detect faint signals without saturating strong signals. Sensitivity and linear range depend on which CCD system you choose.

Even with a digital imager, chemiluminescent Western blot signals are still the result of an enzymatic reaction. The time-dependent enzymatic reaction may still lead to saturation and inaccurate results.

Digital Imaging of Fluorescent Blots

Fluorescent immunoblotting is best performed with near-infrared fluorescent dyes and imaging systems. Background autofluorescence of membranes and biological samples is low in the near-infrared region, enabling high sensitivity. To detect faint signals without saturating strong signals, use an imaging system with a wide linear dynamic range.

Are you experiencing detection system saturation? Find more information about saturation in this full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Do you have a question about your Western blot normalization strategy or how publication requirements for Western blots have been changing? Contact us and let our experts help!

Saturation Limits Accurate Western Blot Normalization

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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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Do you have a question about your Western blot normalization strategy, the importance of replicates. or how publication requirements for Western blots have been changing? Contact us and let our experts help!