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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Chemiluminescent Western Blot Substrate Temperature Affects Signal Strength on Western Blots

The temperature at which a chemiluminescent Western blot substrate is used can affect the strength of the signal that is captured from Western blot images. Really?? Absolutely! This is because enzyme activity is greatly reduced when it is cold. The substrate needs to be equilibrated to room temperature for digital imaging. This is true with film as well, but there may be a period of time after adding substrate and exposing to film during which the substrate has had a chance to equilibrate to room temperature.

In the table below, we show data from an experiment in which we tested the affect of temperature on Western blotting signal. For one blot, SuperSignal® West Pico chemiluminescent substrate was used right out of the refrigerator – cold, 4 °C. For the other blot, the chemiluminescent Western blot substrate was allowed to come to room temperature before digital imaging. As you can see the signal difference is quite large.

Optimal Blot Unsatisfactory Blot
Images Optimal Blot when Substrate is at Room Temperature Unsatisfactory Blot when Substrate is Cold
Conditions:
Substrate SuperSignal® West Pico SuperSignal® West Pico
Substrate at room temperature Substrate cold
Sensitivity Standard Standard
Performance Signal – 1,740 Signal – 200

So make sure your substrate is at room temperature before using, especially when you are imaging with a digital imager!

Here are some other blog posts on 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:

Annotate Visible Protein Ladders on Chemiluminescent Westerns with the WesternSure® Pen

Demonstrating the WesternSure PenIf you doing chemiluminescent Western blots, and are imaging either with film or with a digital imager, the WesternSure™ Pen can be a very useful addition to your experimental process. This newest member of the LI-COR WesternSure chemiluminescent reagent line can be used to annotate visible protein ladders prior to chemiluminescent Western blot detection.

The pen is optimized for detection using the C-DiGit® Blot Scanner or the Odyssey® Fc Imaging System, and is suitable for use with film or other imaging systems. The WesternSure Pen is a unique marker that delivers an ink which emits light when incubated with commonly-used chemiluminescent substrates, including WesternSure PREMIUM Chemiluminescent Substrate. The ink is faintly visible for easy identification of marked membranes.

Here are a few tips to get the best performance from your WesternSure Pen:

  • Lightly touching the pen to the membrane should be enough to transfer ink to the membrane.
  • Do not push down on the nib so hard that it creates an uneven surface on the membrane.
  • Membranes may be annotated when damp after transfer, or when dry.
  • Annotated membranes may be stored dry at ambient temperature or 4 ºC for up to 1 week before starting the Western blot detection process.
  • If ink is not flowing smoothly onto a damp membrane, trace over the band until it is annotated to the desired effect.

Data using the WesternSure PenFigure 1. Chemiluminescent detection of visible protein standards. The WesternSure Pen (LI‑COR P/N 926‑91000) was used to mark the blue protein standards (panel A) for chemiluminescent Western blot detection. The blot was exposed to WesternSure PREMIUM chemiluminescent substrate and imaged on Odyssey Fc Imaging System (panel B).

If you would like some tips on how to troubleshoot chemiluminescent Western blots, read Good Westerns Gone Bad – Maximizing Sensitivity on Chemiluminescent Western Blots.

Good Western Blot Image Signal Acquisition Relies on Uniformly Wet Western Blots

Have you discovered the cause of the weak signals from your chemiluminescent Western blot yet? Well, let’s keep going. Here is another possible cause – the uniform wetness of the blot. It’s important to keep your Western blot membrane uniformly wet during the entire Western blot image acquisition.

Why does this matter? Well, if you don’t add enough substrate, the membrane will not stay wet, and there will be no enzymatic activity. And, that means no signal to detect.

Precaution/Solution:

  • Use more substrate prior to imaging
  • Do not completely blot off all of the substrate before imaging

For C-DiGit® Blot Scanner:

  • Wrap the blot in plastic wrap or cover with a plastic sheet protector
  • Incubate blot with substrate directly on scanner bed

Below is a table showing results of an experiment in which blots of varying degrees of wetness were imaged. You can clearly see that the wet blot and the damp blot give the best results. For both, the blots were protected from drying out by using a 1-ply sheet protector that was placed on top of the blot.

Optimal Blot Optimal Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Wet Blot Optimal Chemiluminescent Damp Blot Unsatisfactory Image
Conditions: Wet blot Damp blot Dry blot
Imaging Method Imaged in 3.0 mL of SuperSignal® West Dura1 substrate placed on the scan bed of the C-DiGit Blot Scanner with 1-ply sheet protector on top. Excess SuperSignal® West Dura1 substrate removed, then imaged on the scan bed of the C-DiGit Blot Scanner with 1-ply sheet protector on top. Blot dried before imaging.
Performance LOD – 640 ng LOD – 640 ng LOD – None detected

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

We still have 5 more possible causes of weak signals in chemiluminescent Western blots to review, so stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!

Watch this short video to see how to correctly place a Western blot on the C-DiGit Blot Scanner surface.

Related posts:

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 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 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
Conditions:
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

    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!