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!

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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?.

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!