April 1, 2014, LINCOLN, NE, USA:
Sensel Raster, of the International Pixel Coalition (IPC), blames an increase in demand from biotech imaging applications. “These fancy scientists think pixels grow on trees!” laments Raster.
Just look at how this pixel shortage is affecting the LI-COR Biotechnology Website!!
Okay, I know, research budget money is tight and you want to make your reagents stretch as far as possible, but it really not a good idea to dilute your chemiluminescent Western blotting substrate.
Why? It’s because the rate of reaction is determined by the ratio of enzyme to substrate. Diluting substrates will dramatically impact the overall generation of light. Then, you will have to repeat the experiment, and you end up using more substrate anyway!
| Optimal Blot | Unsatisfactory Blot | |
|---|---|---|
| Images |  |
 |
| Conditions: | ||
| Substrate | SuperSignal® West Dura1 | SuperSignal® West Dura1 |
| Substrate NOT diluted. | Substrate diluted 1:1 (in water) | |
| Performance | LOD – 1.25 µg | LOD – 2.5 µg |
1Comparable to WesternSure™ PREMIUM Chemiluminescent Substrate
So don’t skimp – use the substrate full strength the first time to ensure that you are seeing all of your protein bands. Or you might just have to repeat the experiment (and that will just cost you more time and money. . .)!
Here are the other nine possible causes of weak chemiluminescent Western blot signals:
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 |  |
 |
| Conditions: | ||
| Substrate | SuperSignal® West Pico1 | SuperSignal® West Pico1 |
| Incubated for 5 minutes | No incubation | |
| Substrate at room temperature | Substrate at room temperature | |
| Performance | LOD – 2.5 µg | LOD – 5 µg |
1Comparable to WesternSure™ ULTRA Chemiluminescent Substrate
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:
iPhone and iPod are all registered trademarks of Apple Inc.
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 |  |
 |
| Conditions: | ||
| Substrate | SuperSignal® West Pico1 | SuperSignal® West Pico1 |
| Substrate at room temperature | Substrate cold | |
| Sensitivity | Standard | Standard |
| Performance | Signal – 1,740 | Signal – 200 |
1Comparable to WesternSure™ ULTRA Chemiluminescent Substrate
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:
Standard and High Sensitivity Settings on the C-DiGit
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 |  |
 |
| 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:
Straight from Cupid for specific bonding!
IRDye® and VRDye™ Secondary Antibodies are the perfect match for your research! We offer highly cross-adsorbed secondary antibodies conjugated to:
To find which LI-COR secondary antibody is the perfect match for your experimental needs, be sure to review the specific applications for which each dye-conjugated secondary antibody is recommended. We also have Protein Labeling Kits in various dye ‘flavors’. Protein labeling kits are cost-effective alternatives to more expensive custom antibody labeling services:
IF, however, you find you do have a special labeling or synthesis need, LI-COR now offers a variety of custom labeling and synthesis services that go beyond our basic offerings. Based on our many years of experience in dye conjugation, our custom services provide a unique solution for most custom needs. We also offer protocol development for In-Cell Western™ Assays, Western blotting, and other applications. The newest offering from our Custom Services group is Reactive Oxygen Species probes.
Happy Valentine’s Day from LI-COR!
If 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:
Figure 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.
In the US, to order the WesternSure Pen online. For order inquiries outside the US, please contact your local sales office.
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 Pico2 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 |  |
 |
 |
| 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 |  |
 |
 |
| 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 |  |
 |
 |
| 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
2Comparable to WesternSure ULTRA Chemiluminescent Substrate
Related posts:
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:
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 |  |
 |
 |
| 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:
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 |  |
 |
 |
| 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 |  |
 |
 |
| 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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