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

westernsure-premium-926-95000Okay, 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
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:

Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals

Standard and High Sensitivity Settings on the C-DiGit
Standard and High Sensitivity Settings on the C-DiGit
Making sure that the sensitivity setting is optimal to capture the most signal from your chemiluminescent Western blot could be the difference between getting a good, strong signal or getting a signal that you can barely see. This is our possible cause 7 for weak chemiluminescent signals.

How can you avoid possible cause 7 for LI-COR chemiluminescent imagers? On the C-DiGit® Blot Scanner, use High Sensitivity setting (12-min scan) for more sensitive detection. On the Odyssey® Fc Dual-Mode Imaging System, use a longer integration time (up to 10 min). Why is this important? Well, digital imaging with the C-DiGit Blot Scanner or Odyssey Fc Imager will not generally reach a saturation point. Begin with a longer acquisition time to ensure best sensitivity, then optimize to shorter scan times.

In Table 1 below, we tested the performance differences of a Western blot detected with SuperSignal® West Dura on the C-DiGit Blot Scanner when the same blot is imaged on High Sensitivity (12 min scan) versus Standard Sensitivity (6 min scan). As you can see, the longer scan time and higher sensitivity make a big difference in the results.

Table 1 Optimal Blot Satisfactory Blot
Images Optimal Sensitivity Setting on C-DiGit Satisfactory Chemiluminescent Western Blot
Conditions: SuperSignal West Dura1 SuperSignal West Dura
Sensitivity High (12 min) Standard (6 min)
Performance Signal – 12,300 Signal – 5,030

1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate

So be sure to check your sensitivity settings before you scan!

Related posts:

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!

Learn more about:

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

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?
  • Is Trying to Get into the Darkroom to Develop Your Western Blot Film Giving You Nightmares?

    See your darkroom nightmares come to life in this short feature presentation from LI-COR. You never know what could be behind your darkroom door . . .

    If you want to avoid future nightmares, check out the C-DiGit® Chemiluminescent Western Blot Scanner.

    Everything you love about film, without the hassles – or the darkroom!!

    Weak Signals on Chemiluminescent Westerns: Possible Cause 2 – Not Enough Substrate

    The amount of chemiluminescent Western blot substrate you use can make a big difference in the results that you get. If you do not add enough substrate to your blot, the light-generating luminol will be depleted, leading to fewer photons (light) being released. (For more information on light collection and the chemiluminescent reaction, read “Imaging Chemiluminescence by Scanning“.)

    Below are the results of an experiment where we looked at the performance differences when incubating the blot in different volumes of SuperSignal® West Pico. The three blots have the same LOD (2.5 μg/well); however, signal intensity varies. Blots were all imaged on the C-DiGit® Blot Scanner. lmages are normalized to the LUT (Lookup Table) of the optimal blot. (Read more about Image Studio™ Software or download FREE Image Studio Lite Western Blot Analysis Software.)

    Optimal Blot Satisfactory Blot Unsatisfactory Blot
    Images Optimal Blot for Substrate Amount Use Chemiluminescent Western showing Satisfactory Results for Substrate Amount Used Unsatisfactory Chemiluminescent Western blot results with low signal
    Substrate SuperSignal West Pico SuperSignal West Pico SuperSignal West Pico
    Substrate Volume 3.0 mL substrate 1.5 mL substrate 0.75 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 Bright signal Moderate signal Low signal

    For more hints and tips, stay tuned to future blog posts.
    Related posts:
    Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction

    Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

    Example of Multiplex Western Blotting using the MPX Blotting SystemMultiplex Western blotting is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

    Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

    • Primary antibodies – to determine the primary antibody that has the right specificity and the right dilution for use
    • Antibody incubation times
    • Blocking conditions – which blocking buffer will give you the optimum results
    • Secondary antibodies – what dilutions is best to use without getting a lot of non-specific binding?
    • Or just about anything else you need to optimize!

    Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

    WesternSure® Chemiluminescent Western Blotting Reagents from LI-COR®

    WesternSure Chemiluminescent Western Blotting ReagentsDetect your Western Blots with Confidence! Use NEW! WesternSure® Chemiluminescent Reagents from LI-COR!

    Now, in addition the great imaging systems for chemiluminescent Western blots, LI-COR Biosciences offers chemiluminescent Western blotting substrates and HRP-conjugated secondary antibodies for use in performing your BEST chemiluminescent Western blots ever. And, the WesternSure Pen is used to annotate visible protein ladders prior to chemiluminescent Western blot detection.

    WesternSure chemiluminescent Western blotting reagents offer the best performance available when compared to other competitive products on the market. WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots.

    WesternSure HRP-conjugated secondary antibodies (Goat anti-Mouse and Goat anti-Rabbit) are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure PREMIUM chemiluminescent substrates.

    Happy Blotting!

    Coming in 2013! LI-COR® C-DiGit® System – Digital Film for Chemiluminescent Western Blot Imaging

    Are you tired of waiting in line for the darkroom? Or spending your precious budget monies on all that film? OR having to do multiple exposures to get just the right image for publication? Then, you need DIGITAL FILM!

    But what, you may be asking, is digital film? Well, that’s the LI-COR C-DiGit System! Image your chemiluminescent Western blot and get great images for publication the FIRST time!

    Which Western Blot Detection Method Should You Use?

    Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which Western blot detection method should you choose? Find out how the three common Western blot detection methods compare to each other in terms of time, sensitivity, and other important factors. The, choose what works best for your research.

    Fluorescent detection: Fluorescent detection uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.

    Enzymatic detection: Chemiluminescent and colorimetric methods use secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP). Signal-generating substrates are used.

    Fluorescent detection uses NIR fluorescent dyes to generate a signal.
    Secondary antibodies are labeled with dyes such as IRDye 800CW or IRDye 680RD
    • Digital imaging reveals target protein signals with high sensitivity
    • Quantitative (signal is proportional to the amount of target protein present)
    • Stable fluorescent signals are stable
    Multiplex detection of multiple protein targets without stripping and re-probing

    Chemiluminescent detection
    uses the horseradish peroxidase (HRP) enzyme and a luminescent substrate.
    • Enzymatic reaction produces light that is detected by film exposure, or digital imaging with CCD camera
    • Multiple exposures typically required to capture optimal signals and avoid signal saturation
    • Very sensitive
    • Cannot be multiplexed
    • May not be quantitative

    Colorimetric detection uses the alkaline phosphatase enzyme.
    • Enzyme converts a soluble chromogenic substrate to a colored, insoluble product that precipitates onto the membrane and produces colored bands
    • Development of the blot is stopped by washing away the soluble substrate
    • Simple and cost-effective
    • Limited sensitivity

    Comparison of Chemiluminescence and Infrared Fluorescence
    Detection for Western Blotting
    Chemiluminescence IR Fluorescence
    Sensitivity +++ +++
    Linear Dynamic Range 10-50 fold >4000 fold
    Multiplex Detection No Yes
    Signal Stability Hours Months – Years
    Enzyme Conjugate HRP
    Substrate Luminol-based None Needed
    Detection/Documentation Film Exposure/Digital Imaging Digital Imaging
    Membrane Compatibility Nitrocellulose or PVDF Nitrocellulose or PVDF