Article Category: C-DiGit System

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:

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:

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.

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?
  • The History of Film. What Does It Tell Us About The Future of Using Film for Western Blot Images?

    small green question markIf you capture Western blot images, chances are you use photographic film and it is fundamental in your daily research activities. But, how much do you really know about it? What IS the future of film use for Western blot imaging?

    Test your knowledge with the questions below:

    Poll Question 1:
    [polldaddy poll=7475861]
    More than 100 years later, Louis Daguerre, a French painter, placed liquid iodine on a silvered copper plate to capture images. He called this process daguerreotyping. Later, in 1875, German physicist, Wilhelm Conrad Rontgen accidentally discovered the X-ray while observing one of these photographic plates.

    Poll Question 2:
    [polldaddy poll=7475874]
    In 1885, George Eastman, an American entrepreneur, invented the first flexible film. Four years later, the first film camera was introduced and KODAK was born.

    Poll Question 3:
    [polldaddy poll=7475881]
    Follow the next five blog posts to learn about how film’s solid foundation and wide use has been interrupted by recent technological developments.

    Related Posts:

    • What is the Future of Film Use for Western Blot Imaging?
    • Here are the answers to the questions. Let’s see how you did.

      • Poll Question 1: white to purple
      • Poll Question 2: his wife’s hand and wedding ring
      • Poll Question 3: 1979

      How did you do? Tweet us your score (3 of 3, 2 of 3, 1 of 3) to @WesternBlotting. We’ll retweet you!

      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

      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!!