Article Category: Odyssey Imaging Systems

Give the Gift of Quantitative Western Blots and Be the Hero in Your Lab this Holiday!

Do you want to be the hero in your lab this holiday season? Watch this video and find out how! (Check out the bloopers at the end of the video!)

Give the gift of quantitative Western blots and your lab will love you for it!

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Happy Holidays from LI-COR! May all your research wishes come true!

Video Infographic: The Fall of Film and Its Effect on Your Western Blots

Watch the video below to see how the past 23 years have contributed to the volatility of the photographic film market, and to show why the availability of film for your Western blots may be at risk.


Solution – Switch to Digital Imaging for Chemiluminescent Western Blots


Solution – Switch to Infrared Detection and Quantitative Western Blots on LI-COR® Odyssey Imagers

Read our previous blog posts to find out the full story behind why the future of film for life science research may be in peril:

The Cost of Film Production May Give Us One Clue Why Film May Not Be Available for Western Blot Imaging in the Future?

Do you know which raw materials are required for producing photographic film? Or, how the changing prices of these goods affect your final cost as a consumer?

The raw materials for film production are some of the world’s most mined natural resources, and thus subject to swinging market prices. Let’s take a closer look at the layers of photographic film and the goods and processes that go into manufacturing the final product. But first, a question:
[polldaddy poll=7597528]
(See the bottom of this post for the answer. :-))

Here is an example of the layers you find in a typical photographic film – the kind you might use for developing Western blots in your lab.
Composition of Film
The top layer, the layer that reacts to light exposure, is the Photosensitive Emulsion Layer. This layer is dull and tacky, and is produced by dissolving silver bars in nitric acid to produce silver halide grains. These photosensitive grains are then suspended and bound in a gelatin solution made from animal hide and bones.

The middle layer, the Film Base, is smooth and shiny. There are three major types of film bases:

  • Cellulose nitrate,
  • Cellulose acetate, and
  • Polyester.

Cellulose nitrate is not commonly used because it is highly flammable. Acetate film was most commonly used between 1920 and 1970. But, because acetate base tends to deteriorate over time and with the invention of polyester, a move toward a new type of film was made in the 1950s. Polyester film, the type primarily used today, is composed from crude oil, or more specifically, petroleum byproducts.

The final layer is the Anti-Halation Layer. This layer prevents halo artifacts from refracted light and is composed of an opaque, heavy color dye. This layer is washed away during processing to reveal a transparent negative, which, in Western blotting, is the final data image.

Stay tuned for more information on how the prices of silver and crude oil affect the prices of film.

Related posts:

Answer to poll question: Yes, photographic film is composed of everything from petroleum to cellulose from animal byproducts. Did you guess correctly?.

What if Film Was No Longer Available? How Would You Capture Your Western Blot Images?

Photographic FilmFilm has been the dominant technology for capturing images for photographers, medical practitioners, and researchers for more than 250 years. Now it’s no longer the sole option. Digital technologies are beginning to impact the future of film. Here’s how and why:

  1. Digital technology is being widely adopted across many different fields including photography, medicine, and scientific research.
  2. The affordability and supply of film has been threatened with the increase of raw material and production costs.
  3. New rules and regulations have been passed in relation to global preservation and green movements.

Because of this, several prominent companies including Kodak and Fujifilm have reevaluated their business initiatives and made decisions regarding the manufacture of certain film-related products.

Get out of the DarkroomIn addition, many universities and institutions are reconsidering their rules and regulations for the disposal and use of hazardous wastes. In general, policies are being made more stringent and punishments for non-compliance more severe. In fact, many new research and medical buildings are being built without darkrooms or the equipment necessary to process film.

Being aware of how these issues, and others, affect the future of film is essential to being able to continue the same quality, or better quality work than you are producing now. Preparing for the future by considering alternative imaging options is becoming more and more essential—especially when processing film comes with additional expenditures and concerns, and requires protocols that rely on toxic chemicals and large amounts of water.

Our next blog post will show you how the cost of raw materials influences the availability and cost of film.

Related Posts:

  • What is the Future of Film Use for Western Blot Imaging?
  • The History of Film. What Does It Tell Us About The Future of Using Film for Western Blot Imaging?
  • What is the Future of Film Use for Western Blot Imaging?

    Western Blot and Hand X-RayX-ray film is a researcher’s Western blotting staple. Chances are you, and many others like you, rely on photographic film to generate results every day. But did you know that the future of this historical technology is being threatened?

    Without warning, several modern day innovations have begun to impact its availability. We’ve gathered the facts, and put together some startling information on the future of film.

    It’s not clear exactly how long we have until photographic film becomes obsolete, but factors such as:

    • product demand
    • the price of raw production materials, and
    • the decisions of manufacturers

    will all have a significant influence on film’s future over the next several years.

    Follow the next six blog posts to learn how the three factors above have and will influence the future of photographic film.

    If you are starting to worry about film going away, find out about digital imaging for chemiluminescent Western blots or infrared fluorescent Western blots!

    Weak Chemiluminescent Western Blot Signals: Possible Cause 3 – Wrong Membrane Placement

    How to Place the Blot on the C-DiGit Blot ScannerSo, we’ve talked about how the substrate rate of reaction can cause weak Western blotting signals and how the amount of substrate used can affect signals on chemiluminescent Western blots. But, there are other possible causes of weak signals.

    The third possible cause of weak signals is the blot membrane placement for imaging on the detection systems, since systems may vary as to how the blot should be placed on the scanning surface. Why is this important? Well, if the blot is placed incorrectly, you may or may not be able to visualize bands. If bands are visualized, they will be substantially reduced in signal.

    As an example, LI-COR has two imaging systems for chemiluminescent Western blots: the Odyssey® Fc Dual-Mode Imaging System and the C-DiGit® Blot Scanner. Blot membrane placement depends on which one you use.

    For the Odyssey Fc Dual-Mode Imaging System, the membrane needs to be placed FACE UP on the imaging tray.

    However, for the C-DiGit Blot Scanner, the membrane needs to be placed on the scanning surface FACE DOWN. (For a quick video demonstrating this, watch “How to Place Your Blot on the C-DiGit Blot Scanner“.) Below is an experiment we did to look at the performance differences between imaging the blot correctly (protein side down) and imaging the blot protein side up on the C-DiGit Scanner. (Images are normalized to the Lookup Table (LUT) of the correctly imaged blot.)

    Correctly Imaged Blot Incorrectly Imaged Blot
    Images Correctly Imaged Chemiluminescent Blot on C-DiGit Scanner Incorrectly Imaged Chemiluminescent Blot on C-DiGit Scanner
    Conditions:
    Substrate SuperSignal® West Dura1 SuperSignal® West Dura1
    Imaging Method Blot imaged protein side facing down Blot imaged protein side facing up
    Performance LOD – 156 ng LOD – 625 ng

    1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.

    For more hints and tips, stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!

    Related posts:
    Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction
    Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate

    Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction

    Optimal Chemiluminescent Western BlotAre you seeing weak signal in your chemiluminescent Western blot data? As we pointed out in a previous blog post, there are 10 possible reasons why this may be happening. Here is the first in our series on the causes and possible solutions/prevention measures you can try to get the best Western blot imaging data you can from your digital imager! We used our Odyssey® Fc Dual-Mode Imaging System and the newest member of our imaging family, the C-DiGit® Blot Scanner, in these studies.

    Possible cause 1: Substrate rate of reaction is not fast enough (e.g., SuperSignal® West Pico)

    Solution: Use WesternSure® PREMIUM or SuperSignal West Femto substrates

    Why this matters: Different substrates have different rates of reaction. Some are developed to give off a lot of light quickly; others give off small amounts of light over longer periods of time. An alternate substrate may be required for digital imaging when imaging blots with low protein abundance.

    Performance differences of three different substrate classifications using C-DiGit® Blot Scanner. All images are normalized to the Lookup Table (LUT) settings of the optimal blot for accurate visual comparison. (Learn more about easy-to-use Image Studio™ Software.)

    Optimal Blot Satisfactory Blot Unsatisfactory Blot
    Images Optimal Chemiluminescent Western Blot Satisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
    Conditions:
    Substrate SuperSignal West Femto SuperSignal West Dura1 SuperSignal West Pico
    Substrate Volume 3.0 mL substrate 3.0 mL substrate 3.0 mL substrate
    Imaging Method
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Scan Setting High High High
    Performance LOD – 78 ng LOD – 312 ng LOD – 2.5 μg

    1Comparable to WesternSure PREMIUM Chemiluminescent Substrate

    If you want to read ahead and find out ways to eliminate or avoid the other 9 causes of weak signals on chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots. Otherwise, stay tuned for more posts right here!

    10 Possible Causes of Weak Signals on Chemiluminescent Western Blot Images

    Weak Signals on Chemiluminescent Western BlotsAre you seeing weaker than expected (hoped for. . .) signal on your chemiluminescent Western blot images with your digital imager? Not sure what could be causing this? Well, here is a list of 10 possible reasons why you might be seeing weak signals in chemiluminescent Western blot data:

    1. The chemiluminescent substrate does not have a fast enough rate of reaction.
    2. Not enough substrate was added to the blot.
    3. Membrane was placed on the detection system incorrectly.
    4. Blot was not detected or processed on the same day it was imaged.
    5. Blot was not kept uniformly wet during the entire image acquisition.
    6. Blot was exposed to film BEFORE imaging on a digital imager.
    7. Blot was imaged using incorrect sensitivity setting (learn about the easy-to-use Image Studio™ Software. Try FREE Image Studio Lite Western Blot Analysis Software to see just how easy it is!)
    8. Chemiluminescent substrate was too cold.
    9. Chemiluminescent substrate was not incubated for 5 minutes.
    10. Substrate was diluted.

    Hum, that’s quite a list! For details on ways to eliminate or avoid these causes and get great results with your chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.

    Rethinking the Traditional Western Blot

    Traditional Western blotting is a labor-intensive process that includes gel electrophoresis, protein transfer to a blotting membrane, incubation with primary and secondary antibodies, and chemiluminescent or fluorescent detection of target proteins. (View a typical Western blotting workflow.) Day-to-day reproducibility is poor, because small variations in lysate preparation, gel loading, electrophoresis, transfer, and detection are unavoidable sources of technical variability.

    Snapshot of In-Cell Western Assay MethodThe In-Cell Western™ (ICW) Assay, a quantitative immunofluorescent method, is an alternative to traditional Western blots that increases both reproducibility and sample throughput. (View a typical ICW workflow.)

    We recently hosted a webinar called “Rethinking the Traditional Western Blot”, during which John Lyssand, PhD, from LI-COR Biosciences, discussed the In-Cell Western Assay and an example of its use in neuroscience research, in this case, Alzheimer’s Disease. The In-Cell Western Assay enables screening and analysis of many more samples in each experiment, eliminates error-prone protocol steps, and delivers higher reproducibility for biological and technical replicates.

    ICW Use: Tau Protein Accumulation and InhibitionThe data presented demonstrated how ICW assays were used in Alzheimer’s Disease research to screen HSP90 inhibitors for their effectiveness in reducing tau activity levels. Dr Lyssand discussed how and why the In-Cell Western Assay is superior to traditional methods for screening of cell samples.

    If you didn’t have a chance to join us in September for “Rethinking the Traditional Western blot”, you can view this webinar online and on-demand. Check out the information on In-Cell Western assays on our website. You can also read Professor Dickey’s white paper as cited above that outlines how he and his group used higher throughput method to study Alzheimer’s Disease.

    Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

    The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

    Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

    Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
    Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
    PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

    The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

    Fluorescent images were acquired at different time intervals after probe injection.
    Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

    Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.


    Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

    AA Chimote, Z Kuras, and L Conforti
    Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
    Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

    In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

    Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.


    Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

    M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
    Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
    Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

    Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

    Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.


    Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

    V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
    Immunotope, Inc., Doylestown, Pennsylvania, USA
    Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

    CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

    Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.


    Check out some of our Publications Lists for: