Article Category: Imaging Systems

Are You Experiencing Detection System Saturation?

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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

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

Reference:
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.

CONCLUSIONS
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

Your Commitment to Producing Reproducible Research is Critical

Sign up for LI-COR’s “Reproducibility in Science Webinar” Series. The first webinar is on March 25, 2015.

Reproducibility is becoming a highly discussed issue in all research sciences. The ability for major research findings to be independently replicated after an initial experiment is essential to building upon foundational discoveries. When experiments are not conducted thoroughly or published articles lack sufficient details for replication, we lose the ability to move ahead with accurate science. This is a major problem for researchers today.

NIH QuoteThis problem will only begin to be addressed if institutions, universities, industry, and others alike take on the responsibility of producing scientific experiments and reporting scientific methods that can be replicated at a later date. Thus, the conventions of reproducible science are paramount to the future of biomedical research findings in particular.

Several areas are being scrutinized in the discussion on biomedical reproducibility. Including:

  • Thoroughness of experimental details in journal articles
  • Review of studies submitted to journals
  • Scientific fraud
  • Utilization of highly reproducible techniques

reproducibility initiative logo smallThoroughness in research is important, because without knowing all the details of a foundational experiment future scientists are unable to efficiently build upon that research. To increase thoroughness, the Reproducibility Initiative, headed by Elizabeth Iorns, is advising full disclosure of experimental procedures in published papers. The initiative aims to identify and reward high quality, published research that can be successfully reproduced by independent validation labs. The first step in this process is pinpointing a pool of research that is true and accurate —a task The Reproducibility Initiative has begun by investigating 50 of the most impactful cancer biology studies from 2010 – 2012.

In light of the growing concern regarding scientific reproducibility, the review processes for scientific journal submissions are seeing stringent changes as well. The plans to increase the reproducibility of published papers laid out by the National Institutes of Health (NIH) at the beginning of the year are just one example. In their plan the NIH instituted a training module for enhancing the transparency of cited methods, provided a checklist for routine evaluation of grant applications, and began to urge scientific journals to revise their current review practices. Since then, high-impact journals like Nature and Science have implemented precautionary statistical checklists intended to qualify submitted research papers before publishing them in their magazines.

Unfortunately, though, there are times reported science is proved to be inaccurate, and fraudulent papers claiming breakthrough research are retracted. These retractions can severely affect scientists who have based their careers on such published inaccuracies.

ireland flag smallIn response, Ireland has taken precautions against fraudulent publication. By the end of the year The Science Foundation Ireland will be funding auditors at leading universities. The auditors will look into best practices related to research, procedures “for reporting and investigating misconduct; whether management has followed those procedures in real cases; and whether any investigations have been carried out to a satisfactory standard.” The purpose of these audits is to encourage researchers to take protocols seriously and to put standards in place that will decrease the likelihood of scientific fraud occurring.

Another area of the reproducibility discussion highlights the need for highly consistent research techniques and instrumentation. The nature of complex research and varying protocols between labs can cause inherent fluctuating results from experiment to experiment. To help combat the variability, there is a need for improved and consistent training of researchers using Western blotting and other scientific techniques in their research, just as there is a need for the instruments researchers use to be of the highest quality and to generate reproducible results. Putting more emphasis on training researchers and utilizing the highest quality instruments will help to improve the reproducibility of the studies research labs are currently conducting.

Only time will tell if the scientific community will really begin to take the issues and repercussions of reproducible science seriously. While science is shifting it is important you stay ahead of the curve and close the gaps in your research confidently. Your commitment to producing reproducible research is critical to redressing the reputation of the scientific method from beginning research stages to the published piece.

Are your findings reproducible? Read more about how reproducibility is affecting the life sciences and where the future of Western blotting may be headed.

If you’d like to learn more about reliable instrumentation, check out LI-COR Imaging Systems, which offer a digital imaging solution that ensures reproducible results. See how LI-COR can help you improve your research.

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.

Register to win a C-DiGit® Chemiluminescent Western Blot Scanner today.

Is Research Funding an Issue in Your Lab?

Note: Currently, the SURG program is available in Austria, Denmark, Finland, France, Germany, Iceland, Ireland, Norway, Puerto Rico, Sweden, Switzerland, the United Kindgom, and the United States.

NIH Funding Graph smallerIs research funding a main concern at your institution? In a study of 3700 researchers by the American Society for Biochemistry and Molecular Biology, “68% of respondents do not have the funds to expand their research operations.” Furthermore, “65% of respondents have had difficulties receiving funding.” This is an alarming number for the research community today.

Funding has been on the decline for some time now (see chart below), especially after the 2008 recession and the NIH sequester in 2013. In 2013, the NIH handed out “approximately 640 fewer research project grants compared to FY 2012.”

As budgets are tightened across the board, funding in general may be an issue in your lab. Besides funding to back research projects, faculty and researchers need reliable instrumentation in their labs to ensure reproducible, consistent results.

How will your institution remain equipped in an ever-increasing competitive environment? The LI-COR SURG Program** could help. The SURG – Science Undergraduate Research Grant – Program is designed for faculty researchers and their students to gain access to cutting edge life science technology. If students are learning Western blotting or gel imaging techniques, this grant program could be a perfect fit.

Odyssey Fc smallerLI-COR SURG grants are a 40% match from LI-COR. The process takes ten minutes to apply.

There’s no guarantee funding will increase in the future. This program could help ensure your research is supported by superior digital imaging technology. Check out the SURG Program** offered by LI-COR Biosciences if you’re interested in learning more. Here’s more information on the Odyssey® Fc Imaging System – LI-COR’s digital imaging solution offered through the SURG program.

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.

Reference:

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

URGENT MESSAGE: Global Sources Report Imminent Pixel Shortage!

Global pixel manufacturers report that a pixel shortage has reached crisis proportions.

Global Pixel Shortage
April 1, 2014, LINCOLN, NE, USA:

Sensel Raster, of the International Pixel Coalition (IPC), blames an increase in demand from biotech imaging applications. “These fancy scientists think pixels grow on trees!” laments Raster.

Just look at how this pixel shortage is affecting the LI-COR Biotechnology Website!!
April Fools Day Home Page

Learn more about this crisis.

Possible Cause 10 for Weak Chemiluminescent Western Blot Signals: Diluting Substrates

WesternSure Chemiluminescent Western Blot ReagentsOkay, I know, research budget money is tight and you want to make your reagents stretch as far as possible, but it really not a good idea to dilute your chemiluminescent Western blotting substrate.

Why? It’s because the rate of reaction is determined by the ratio of enzyme to substrate. Diluting substrates will dramatically impact the overall generation of light. Then, you will have to repeat the experiment, and you end up using more substrate anyway!

Optimal Blot Unsatisfactory Blot
Images Optimal Western Blot - Substrate Not Diluted Unsatisfactory Chemiluminescent Western Blot - Substrate Diluted
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal® West Dura1
Substrate NOT diluted. Substrate diluted 1:1 (in water)
Performance LOD – 1.25 µg LOD – 2.5 µg

1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate

So don’t skimp – use the substrate full strength the first time to ensure that you are seeing all of your protein bands. Or you might just have to repeat the experiment (and that will just cost you more time and money. . .)!

Here are the other nine possible causes of weak chemiluminescent Western blot signals:

Don’t Rush Substrate Incubation Time for Chemiluminescent Western Blots

Substrate Incubation Time is Important!Five minutes can seem like a long time, especially when you are waiting to image your chemiluminescent Western blot. But it is really important that you follow the manufacturer’s recommendation for incubation time. Typically, this is five (5) minutes for optimal photon emission – for both film and digital imaging.

So, set the timer for 5 minutes, grab your iPhone® or iPod® – or the crossword, and relax until the buzzer goes off.

To test this, we imaged a chemiluminescent Western blot immediately after adding the chemiluminescent substrate and then imaged a blot where we waited 5 minutes – answered a few emails, looked at the news, and downloaded a new app – and THEN imaged the Western blot. As you can see, incubating allowed us to see more bands and gave much better Western blotting results.

Optimal Blot Unsatisfactory Blot
Images Optimal Blot - 5 Min Substrate Incubation Unsatisfactory Blot - No Incubation
Conditions:
Substrate SuperSignal® West Pico SuperSignal® West Pico
Incubated for 5 minutes No incubation
Substrate at room temperature Substrate at room temperature
Performance LOD – 2.5 µg LOD – 5 µg

So slow down, take a breath, and wait for your chemiluminescent Western blot substrate to incubate on your Western blot before imaging.

Here are some other blog posts on possible causes of weak chemiluminescent Western blot signals:

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