Article Category: Imaging Systems

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.

If Comparing Film and Digital Imagers, Expose Blot on Digital Imager First.

If you are trying to compare how the same chemiluminescent Western blot looks when imaged on a digital imager (like the C-DiGit® Blot Scanner) with how it will look when imaged on film, it’s important to know that you should expose the blot to film BEFORE imaging on a digital imager.

Why does this matter? Digital imaging requires capturing the most photons being generated, which is typically immediately after a 5-minute chemiluminescent substrate incubation. Time may be more of an issue with some substrates. For more information on how film and digial imaging compare, read Western Blot Analysis: Comparison of film and the C-DiGit Blot Scanner.

In Table 1 below, performance differences of a Western blot detected with SuperSignal® West Pico when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Blot with SuperSignal West Pico Unsatisfactory Blot with West Pico Unsatisfactory Blot with West Femto
Conditions: Immediately after incubation with SuperSignal West Pico 26 min after incubation 51 min after incubation
Imaging Time Immediately after incubation with SuperSignal® West Pico 26 min after incubation 51 min after incubation
Scan Setting High High High
Performance LOD – 625 ng, Signal – 338 LOD – 625 ng, Signal – 114 LOD – 625 ng, Signal – 32.2

In Table 2, Performance differences of a Western Blot detected with SuperSignal West Dura1 when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are normalized to the LUT of the optimal blot.

Table 2 Optimal Blot Satisfactory Blot Satisfactory Blot
Images Optimal with West Dura Satisfactory with West Dura Satisfactory with West Dura
Conditions:
Imaging Time Immediately after incubation with SuperSignal West Dura 24 min after incubation 48 min after incubation
Scan Setting High High High
Performance LOD – 156 ng, Signal – 12,300 LOD – 156 ng, Signal – 10,400 LOD – 156 ng, Signal – 9,090

In Table 3, Performance differences of a Western Blot detected with SuperSignal West Femto when the same blot is imaged over time. Blot was incubated 5 min in substrate before imaging on the C-DiGit Blot Scanner. Images are linked to the LUT of the optimal blot.

Table 3 Optimal Blot Satisfactory Blot Satisfactory Blot
Images Optimal Blot with West Femto Satisfactory Blot with West Femto Satisfactory Blot with West Femto
Conditions:
Imaging Time Immediately after incubation with SuperSignal West Femto 24 min after incubation 48 min after incubation
Scan Setting High High High
Performance LOD – 156 ng, Signal – 11,500 LOD – 156 ng, Signal – 8,120 LOD – 156 ng, Signal – 6,860

1Comparable to WesternSure® PREMIUM Chemiluminescent Substrate

Related posts:

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.

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Troubleshooting Chemiluminescent Western Blots: Possible Cause 4 for Weak Signals – Blot Processing

Sometimes life in the lab gets crazy, right? You are finishing a Western blot and you realize that you are supposed to be at an important lecture across campus in 10 min!! Or, your spouse calls to say that one of the kids needs to be picked up as soon as possible. Yikes! The challenge is that blots should be processed and detected on the same day. And, the secondary antibody should be incubated the day of imaging and fresh substrate added just before imaging. Is it that important to your results? Yes, it is and just to prove it, we did a few experiments.

In Table 1, we studied performance differences when the same blot is imaged immediately after processing vs. stored overnight dry and then imaged. In Table 2, we looked at performance differences when the same blot is imaged immediately after processing vs. stored overnight wet and then imaged. Blots in both tables were all imaged on the C-DiGit® Blot Scanner. (And, all images are normalized to the Lookup Tables (LUT) of the respective optimal blot.)

For both experiments, you can see that saving the blot to image the next day is not a very good choice. This is because the secondary antibody and/or the chemiluminescent Western blot substrate is not stable enough for acceptable photon emission when digitally images after the day it is applied.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Processing Time Same Day Next Day Next Day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight dry, at room temperature Blot stored overnight dry, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg
Table 2 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Process Time Same day Next day Next day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight wet in PBS, at room temperature Blot stored overnight wet in PBS, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg

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:

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!

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:

Cost of Film Raw Materials Keeps Rising. . .Is Western Blot Film Imaging Worth the Cost?

Film Imaging Examples for Photography, Dentistry, Medicine, and ResearchAs the cost of raw materials for medical x-ray film manufacture rises, so does the cost of a box of film.

So, how has the price of raw materials changed over time?

Basically, the cost of

  • Silver has increased nearly 10-fold in the last decade.
  • Crude oil has tripled in the last decade.

In turn, this has caused several successive price increases to be passed along to the consumer – you.

Rising costs of manufacturing filmNot only is the cost of materials affecting the future of the film you use every day, but it’s also having a dramatic effect on the move to digital imagery in other industries, including:

  • photography,
  • dentistry,
  • and medicine.

The most dramatic influence has been in the photography field. The quick movement from film to digital imagery in this arena has influenced decisions about the use of film in the dentistry and medical industries, which in turn has effected the decisions of manufacturers to continue providing a product that is seeing a steady decrease in demand.

Check back on this blog for an infographic detailing the rising costs of raw materials and the rising costs of film. Also, see how the changes in market trends have affected some of the biggest film manufactures worldwide.

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.

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