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

    Create a Work Area and Import an Image into this Free Western Blot Analysis Software

    In this series of posts, you will discover how easy it is to use Image Studio™ Lite. This free Western blot analysis software from LI-COR® allows you to easily create your own work area and then import images from numerous sources. So, if you have an old film scan, or an image from another chemiluminescent Western blot imaging system, try using this free Western blot analysis software. In subsequent posts, we will talk about the other functionalities of Image Studio Lite.

    The first video in this post will show you how to open Image Studio Lite software and create a work area. (If you need to find out the installation requirements for Image Studio Lite, see the 17-Jul-13 Post, Computer Requirements to Get Started with Free LI-COR® Image Studio Lite Software).

    This second video walks through the steps needed to import an image into Image Studio Lite. Image Studio Lite analyzes images in the tif, png, or jpeg format as well as images acquired with past versions of the Odyssey® or Pearl® imaging systems software.

    Download FREE Image Studio Lite Western Blot Analysis Software from LI-COR and get started right away! (Bonus points for telling your friends about it too! :-))

    Image Studio Lite – A Dream Come True without the Unicorn!

    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!

    No Darkroom? No Problem! Get the LI-COR® C-DiGit® Western Blot Scanner !

    C-DiGit Blot Scanner - a Compact Personal Chemiluminescent Imaging System
    Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?

    FINALLY, image at your convenience. Keep your C-DiGit Blot Scanner on your lab bench, at your desk, or anywhere you choose (look left and see just how small and portable the C-DiGit Western Blot Scanner really is!). It can truly be YOUR personal chemiluminescent Western imager!

    The C-DiGit Chemiluminescent Western Blot Scanner maintains the simplicity of film exposures without the mess of the darkroom. You perform all of the same steps, without buying film and spending time in the darkroom. The C-DiGit Scanner gives you a complete digital replacement for film – keeping the advantages of film and eliminating many of the drawbacks – saving you time AND money!

    Watch this short video and then visit our website to get your very own C-DiGit Blot Scanner!

    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

    Digital Western Blots – Chemiluminescent Substrate Selection is Critical

    Chemiluminescent Western BlotSo, you’ve selected your primary antibodies with care (see Know Thy Primary Antibody), and you’re using a great HRP-conjugated secondary antibodies. NOW – what about the chemiluminescent substrate?

    Yes, I know, there are tons of different substrates and vendors out there – all claiming to be the best, right? So, how do you choose the right one for your chemiluminescence Western blot?

    One thing to keep in mind is that in this wide variety of chemiluminescent substrates for HRP detection, there are some that are better suited for digital Western imaging than others. In general, choose a substrate with a faster rate of reaction for use with the Odyssey Fc Dual-Mode Chemiluminescent and IR Fluorescent Imaging System or other digital chemiluminescent imaging systems.

    Some substrates that are designed for optimal performance on film may not be suitable for detection on a CCD-based imaging system. Try different substrates to find the one that gives the most desirable image. As you can see from the images below, the substrate you pick DOES make a difference! So choose carefully!

    In the three images below, two-fold serial dilutions of HRP-conjugated secondary antibody (1 ng-1.25 fg) were spotted onto nitrocellulose using a slot blot apparatus. Blots were detected with various chemiluminescent substrates.

    Chemiluminescent Substrate Comparison

    Here are 2 documents with more troubleshooting information:

    Updated February 18, 2015.

    Love Traditional Western Blotting with Chemiluminescence Detection? Go Digital!

    Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging SystemTired of the darkroom being down or the expense of the development chemicals? Oh, AND all that film you go through because you have to do multiple exposures to get the image just right? Go DIGITAL with the Odyssey® Fc Dual-Mode Imaging System.

    Dual-mode? Yes! The Odyssey Fc provides the ability to streamline your chemiluminescent Western blot imaging – no film, no darkroom, just clear fast blot images.

    PLUS this digital imaging system includes two near-infrared fluorescent channels for sensitive, quantitative infrared Western blot imaging. So use ECL or whatever chemiluminescent substrate you usually use but eliminate film (more on best substrates to use in a later post). See how much money you will save going digital!

    Schematic of Digital Chemiluminescent Imaging on the Odyssey Fc

    The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. Learn about FieldBrite™ XT2 technology and the advantages it gives you with the Odyssey Fc.

    The Odyssey Fc Imaging System can also be used for Coomassie-stained protein gel imaging and DNA gel imaging of either ethidium bromide-stained gels or SYTO® 60 stained gels.


    Chemiluminescent Western Blots – FAQs about Primary and Secondary Antibodies

    We’ve discussed some hints on how there can be considerable difference in primary antibodies – so “Know thy Primary Antibody.

    In addition, we’ve received some questions that are frequently asked – hence called frequently asked questions or FAQs – about primary and secondary antibodies when doing chemiluminescent Western blots. So here they are. I am sure there are more. . .so send them our way by commenting on this post!

    Why is the signal missing in the middle of the bands?
    Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.

    Does it matter where I purchased the HRP-conjugated secondary antibodies?
    The reactivity of secondary antibodies ranges widely between vendors. As well, the ratio of HRP enzyme to antibody varies, and may affect the detection of the target. If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.

    Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
    Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.

    What questions do you have?

    Optimizing Chemiluminescent Western blots Technical Note might be a good place to start to get some of those questions answered. And remember you save time and money by going digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!

    Happy Blotting!

    Select the Primary Antibodies You Use with Care for Western Blot Success

    “Know Thyself – and Thy Primary Antibody!”

    Okay, so you’ve done your experiments, run your sample on a gel, and transferred the proteins to a membrane. Now, you need to see if you can detect the protein, what happened to it, how much is there, etc.

    After you block (remember we talked about how important the right blocker is), you will probe with a primary antibody (that is, an antibody produced to detect a specific antigen) to see your molecule of interest. Now, primary antibodies can be produced in a wide variety of species such as mouse, rabbit, goat, chicken, rat, guinea pig, human, etc. There are lots of suppliers of antibodies out there, so it is important to realize primary antibodies for the same antigen can perform very differently. It may be necessary to test multiple primary antibodies for the best performance in your Western blot system.

    In the images below, you can see how different primary antibodies to the same target may react. Serial dilutions of NIH/3T3 lysate were probed with Akt monoclonal primary antibodies from three different vendors. All blots were blocked with 5% skim milk and detected with HRP-conjugated Goat Anti-Mouse and SuperSignal® West Dura chemiluminescent substrate. Western blots were imaged on the Odyssey Fc Chemi channel for 2 minutes, shown with normalized image display settings. You can see that the primary antibodies varied quite a bit. The number and intensity of bands you can detect and the amount of non-specific binding that occurs are definitely different for each one.

    So, take a cue from ancient Greece and get to know your Primary Antibody by doing some testing and optimization.

    The result = GREAT Western Blots!