Article Category: Western Blotting

Are You Experiencing Detection System Saturation?

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.

Western Blot - fig1-detection
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

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.

Blog post 4 - direct-indirect

Requirements for Internal Loading Controls

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

Western blots are packed with potential sources of variability. Variability that isn’t accounted for limits reproducibility and threatens your chances for publication-quality data. Normalization corrects for variability introduced during the process of Western blotting.

So what should you do to get more reproducible data? Use an internal loading control for each blot. Internal loading controls are endogenous sample proteins that are stably expressed and unaffected by experimental conditions.

Requirements for an Effective Internal Loading Control:

  • Linear, proportional response. Signal intensity of the internal control should accurately reflect sample con¬centration and abundance of loading control over a wide range.
  • Low biological variability. Your experimental treatments should not affect the expression of your internal loading control. For example, expression of some housekeeping proteins may vary in response to experimental conditions.
  • Corrects for variation at all stages of immunoblotting. Your internal control should correct for variation that occurs throughout the Western blot process, including gel loading and transfer.
  • Compatible with immunodetection. The strategy you choose shouldn’t interfere with effective down¬stream detection of your target proteins.

For more information about internal loading controls, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

What Factors Affect Normalization?

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

Do you know what factors affect normalization? Routine steps in the Western blotting process such as sample preparation, sample loading, and the detection of multiple proteins can introduce unwanted variability. You should plan to reduce error in every step of the Western blotting process. Without planning, you might get pseudo-quantitative results that don’t reflect the biology of your samples.

Sample Preparation

Blog - Sample prepThe way you prepare your samples can significantly change the results of your experiment. Even small changes in plating, cell lysis, reagent volume, and other technical details can have a surprising impact.

For example, how you lyse your cells affects protein extraction, solubilization, and modification status. The insoluble fraction may retain relevant proteins, affecting your quantitative analysis. Some experimental treatments shift fractions between soluble and insoluble.

For these reasons, it’s important to be consistent when preparing your samples. It’s also good practice to estimate the total protein concentration of each sample after preparation. Bradford, BCA, and Lowry assays are widely used to estimate the total protein concentration. Then it’s possible to adjust gel loading to the estimated protein concentration.

Sample Loading

Blog - sample loadingOverloaded gels create problems. Although strong bands may appear similar, the bands could be saturating either the membrane capacity or the dynamic range of detection. To avoid saturation and inaccurate results, run a standard curve with two-fold serial dilutions of cell lysate. You can then identify the linear range for each target protein.

Detection of Multiple Proteins

You may need to detect multiple proteins to compare relative protein levels, especially if you’re using a housekeeping protein or signaling protein to normalize. Stripping and reprobing is often used to compare different proteins on the same blot, but it can introduce error. Leftover antibodies from incomplete stripping result in artifacts. Overly harsh stripping may result in a loss of sample proteins from the membrane.

If, however, you use near-infrared fluorescent detection, there’s no need to strip and reprobe. Multiplexing is when you detect two different proteins with spectrally-distinct secondary antibodies. Multiplexing is convenient and saves time. It is also more accurate than stripping and reprobing, because no artifacts are introduced, and there’s no possibility for protein loss. With multiplexing, you can use co-migrating proteins, as well as easily identify antibody cross-reactivity.

For more details about factors that affect normalization, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

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

Are Western Blot Results Misrepresented by Film and Photochemistry?

Although most researchers have used film to document Western blots, many may be unfamiliar with the photochemical process that creates a visible image on a sheet of x-ray film. Because this process affects data output, it is important to understand how chemiluminescent signals are recorded by film – particularly if the results will be quantified by densitometry.1

What happens when you expose a Western blot to film?

X-ray film is coated with a photographic emulsion that contains light-sensitive silver grains. Photons of light from the chemiluminescent reaction activate individual silver grains, which are then converted to black metallic silver to create a visible film image. Within the film’s linear response range, your results are proportional to light intensity and duration; this is called the Reciprocity Law.

What goes wrong during film exposure?

Film’s linear response range is extremely narrow (1.0 – 1.5 logs). Above and below that narrow range, “reciprocity failure” occurs – and your bands won’t be proportional to the light produced by the chemiluminescent reaction. It’s important to know that both strong and faint signals are not accurately detected by film, which compromises the accuracy of your densitometry results.
film vs photchem image

How does reciprocity failure affect your densitometry and data analysis?

In this example, film response is only linear between 0.1 ng and 1.56 ng. Above 1.56 ng, bands visually appear stronger on film, but signals are not accurately recorded due to high intensity reciprocity failure.

film vs photochem image 2
RESULT: Strong bands are underestimated by densitometry. Film’s limited dynamic range interferes with accurate detection of strong signals.

Improve the accuracy of your results

The photochemistry of film causes a non-linear response of film to faint and strong signals (reciprocity failure). Saturation of strong signals and under-representation of faint signals means that accurate densitometry is severely limited by film’s shortcomings. When you switch to a digital imager, you will get more accurate results. Read the full paper to learn about all the variables that affect accurate quantification:

  • Enzyme/substrate kinetics and changes in substrate availability
  • Limitations of film exposure and digitization methods
  • Difficulty determining the saturation point of strong signals

Read the full study: Chemiluminescent Westerns: How film and photochemistry affect experimental results

1. Baskin, DG and WL Stahl. Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P.
41(12):1767-76 (1993).

Is Your Chemiluminescent Western Blot Imaging Method a Source of Error and Variability?

Chemiluminescence is a dynamic, enzymatic process that introduces variability and error in your Western blot experiments. It’s often difficult to find the “best” exposure, and the need for multiple exposures limits the reproducibility of your results.

Variability and error are introduced because:

  • Chemiluminescent reaction changes constantly.
    The “best” exposure time is a moving target, so you must optimize and double-check every experiment.
  • Multiple exposures are required.
    Common detection methods cannot accurately capture both faint and strong signals at once, without signal saturation.


Usable Data for Each Detection Method

Film Imager B Odyssey® Fc Imager
film usable range imager b usable range odyssey fc usable range
RESULT: Exposure time dramatically affects data output. Multiple exposures are required to detect strong and faint signals. Signal saturation cannot be determined visually. RESULT: Multiple exposures are required to capture the full range of data. Strong signals are saturated (shown in blue). RESULT: Multiple exposures are not required, because all exposure times yield consistent results. All data are captured in a single exposure without saturation.

In the figure above, film was compared with a conventional, commercially-available CCD imager (Imager B), and the Odyssey Fc imager. To eliminate variability introduced by blotting and chemiluminescent detection chemistry, a Harta luminometer reference plate (standardized light source) was used in place of a Western blot.

The Odyssey Fc imager outperformed both film and Imager B. All signals, from faintest to strongest, were detected – regardless of exposure time in a single exposure. No signal saturation occurred and all signals could be quantified. With film and Imager B, however, longer exposures are needed to detect faint signals. In addition, stronger signals become saturated and cannot be quantified.

Choosing the Odyssey Fc Imaging System as your imaging method reduces variability and error in chemiluminescent Western blotting by giving you:

  • All your data in a single exposure
  • More reproducible results
  • Simplified data analysis

Read the full study to learn:

  • How chemiluminescence detection introduces variability and error
  • How you can improve the reproducibility of your Western blot data

Film and CCD Imaging of Western Blots: Exposure Time, Signal Saturation, and Linear Dynamic Range

The Way Medical Film’s Future is Headed Will Keep You Up at Night

What is the future of medical film?

Film Imaging Examples for Photography, Dentistry, Medicine, and ResearchNearly a year ago we told you why film’s future availability and affordability are in jeopardy. Today, we are still seeing a decreased demand and reduced production volume of film. But there are additional concerns. The environment is suffering because of the hazardous chemical and medical waste produced from using film.

Here are some realities facing Western blotters who use medical film:

  • The federal Resource Conservation and Recovery Act (RCRA) sets regulations for hazardous waste handling and storage.
  • The RCRA has strict laws with authority from the EPA enforcing toxic chemical cleanup.
  • Developer solutions must be neutralized and flushed with large quantities of water to the sewer system.
  • Film sheets should be collected for silver recycling because silver is too toxic to go in landfills.

What are the implications?

stas quoteAs environmental concerns rise and the supply of film is threatened, the sustainability and future of film production are at risk. As a responsible research scientist, you are aware there are environmental considerations and financial incentives for ceasing film use and switching to digital imaging. Read about one researcher who has come to that realization.

What can you do?

Consider an environmentally-friendly Western blot imaging alternative, and:

  • Eliminate your use of medical film
  • Decrease your environmental impact
  • Implement a more sustainable Western blotting technique

c-digit small

Go to bed at night without worrying if you can afford your next box of film or if you are complying with environmental hazardous waste disposal regulations. Go digital.

Studying Colon Cancer? Use the C-DiGit® Scanner for Western Blots.

Cortactin (CTTN) is a substrate of Src tyrosine kinase involved in actin dynamics, and is overexpressed in several cancers. Phosphorylated cortactin (pTyr421) is required for cancer cell motility and invasion. This study demonstrates elevated expression of pTyr421-CTTN in primary colorectal tumors, with no change in mRNA levels. Curcumin (a natural compound derived from the spice turmeric) reduced association of CTTN with plasma membrane fractions in surface biotinylation, mass spectrometry, and Western blot experiments. Curcumin also decreased pTyr421-CTTN levels in certain cell lines.

Western blot analysis of cortactin, actin and GAPDH proteins

Figure 1. Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p<0.05 DMSO vs. curcumin; Student’s T-test. All images are representative of three independent experiments.

Quantitative chemiluminescent Westerns (using the LI-COR® C-DiGit Blot Scanner and SuperSignal® West Pico substrate) showed that curcumin treatment reduced CTTN levels in cytoskeletal fractions, and increased cytoplasmic localization. In Western blotting and immunofluorescent microscopy studies, curcumin induced dephosphorylation of cortactin by activation of the PTPN1 protein tyrosine phosphatase. Western blotting demonstrated that biotinylated curcumin directly binds to PTPN1, and that curcumin blocks the interaction between CTTN and p120 catenin. Curcumin inhibits cell migration in colon cancer cells overexpressing CTTN, and it holds promise as a colon cancer therapeutic.


pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1)
VM Radhakrishnan, P Kojs, G Young, R Ramalingam, B Jagadish, EA Mash, JD Martinez, FK Ghishan, PR Kiela
University of Arizona Health Sciences Center, Tucson, Arizona; Arizona Cancer Center, Tucson, AZ, USA
PLoS ONE 9(1): e85796 (2014). 10.1371/journal.pone.0085796

Avoid Milk Blocking Buffer – Use NEW! Odyssey® Blocking Buffer (TBS)

Odyssey Blocking Buffer (TBS)

In previous posts, we’ve talked about Western blot blocking buffers and how important it is to optimize your blocking conditions to get the best results. As many of Western blot users do, you may just routinely use homemade TBS-milk blocking buffer. It’s inexpensive, and it does the job. . . well, most of the time. . .

What you may not know is using milk blocking buffer can cause issues with certain targets. This may give you the wrong information about the presence or the amount of your target. One good way to determine which blocking buffer system to use is to check to see what the primary antibody vendor recommends. Most recommend TBS-based buffer systems. If the primary antibody requires a TBS-based buffer system, we recommend new Odyssey® Blocking Buffer (TBS).

When should you avoid milk blocking buffer?

  • When using anti-goat secondary antibodies.
    • Reason: Milk contains bovine IgG. Anti-goat secondary antibodies may recognize bovine IgG, resulting in high background.
  • When detecting phosphorylated proteins.
    • Reason: Milk contains phosphorylated proteins, which may result in low to no signal and high background.
  • When using streptavidin-biotin detection systems.
    • Reason: Milk contains endogenous levels of biotin. Streptavidin will detect this, resulting in high background.

OBB TBS and milkHere are the results of an experiment evaluating the use of milk and Odyssey Blocking Buffer (TBS). As you can see, milk masked the detection of this protein and is not a good blocking buffer choice.

Figure 1. Effect of various blocking agents on detection of pAkt and total Akt in Jurkat lysate after stimulation by calyculin A. Total and phosphorylated Akt were detected in calyculin A-stimulated (+) and non-stimulated (-) Jurkat lysate at 10 µg; 5 µg; and 2.5 µg/well. Blots were probed with pAkt Rabbit mAb (Santa Cruz P/N sc‑135650) and Akt mAb (CST P/N 2967) and detected with IRDye® 800CW Goat anti-Rabbit IgG (LI‑COR P/N 926-32211) and IRDye 680RD Goat anti-Mouse IgG (LI‑COR P/N 926‑68070); scanned on Odyssey® CLx (auto scan 700 & 800). pAkt (green) is only detected with Odyssey Blocking Buffer (TBS).

So be sure to optimize your Western blot blocking conditions! The time you spend finding the best blocker will be worth it – and save you from making the wrong conclusions about your experimental data in the future.