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Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals

Posted on February 18, 2014 by licor-admin

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
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!

Find Your Perfect Match with IRDye^® and VRDye™ Secondary Antibodies!

Posted on February 14, 2014 by licor-admin

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:

IRDye 800CW
- Including IgG₁, IgG_2a, and IgG_2b Subclass Specific and Goat anti-Mouse IgM (μ chain specific) Secondaries
IRDye 680RD
- Including Goat anti-Mouse IgM (μ chain specific) Secondary Antibody
IRDye 680LT
- Including IgG₁, IgG_2a, and IgG_2b Subclass Specific and Goat anti-Mouse IgM (μ chain specific) Secondaries
IRDye 650
VRDye 549
VRDye 490

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!

Posted in IRDye Reagents, Reagents | Tagged irdye 650 antibodies, irdye 680lt antibodies, irdye 680rd secondary antibodies, irdye 800cw antibodies, irdye custom labeling, IRDye secondary antibodies, li-cor custom services, licor custom labeling, licor custom services, licor protocol development, perfect match, protein labeling kits, secondary antibodies, secondary conjugates, valentines day, vrdye 490 secondary antibodies, vrdye 549 conjugates, vrdye custom labeling, vrdye secondaries, vrdye secondary antibodies, vrdye secondary antibody conjugates | Leave a comment

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

Posted on February 12, 2014 by licor-admin

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:

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.

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.

Posted in C-DiGit System, Odyssey Imaging Systems, Reagents, Western Blotting | Tagged annotate protein ladders, chemiluminescent western blots, li-cor protein marker, licor protein marker, licor western blot marker, protein ladder marker, protein molecular weight marker, visible protein ladders, visible protein markers, western blot annotation, western blot marker, western blot pen, western blotting marker, western blotting pen, westernsure reagents | Leave a comment

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

Posted on February 10, 2014 by licor-admin

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

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

Posted on January 30, 2014 by licor-admin

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
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.

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

Posted on January 7, 2014 by licor-admin

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!

Give the Gift of Quantitative Western Blots and Be the Hero in Your Lab this Holiday!

Posted on December 18, 2013 by licor-admin

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:

Odyssey CLx Infrared Imaging System – the standard in quantitative Western blots with over 6000 peer-reviewed journal references citing its use in all areas of scientific research
Quantitative Western blotting with near-infrared fluorescent Westerns

Happy Holidays from LI-COR! May all your research wishes come true!

Posted in Odyssey Imaging Systems, Western Blotting | Tagged fun lab video, holiday giving, holiday video with bloopers, odyssey clx, quantitative western blots, science humor, science lab funny video, western blotting | Leave a comment

Video Infographic: The Fall of Film and Its Effect on Your Western Blots

Posted on December 12, 2013 by licor-admin

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:

Posted in C-DiGit System, Odyssey Imaging Systems, Western Blotting | Tagged digital imaging for western blots, digital imaging for westerns, film for western blot imaging, future of film infographic, licor solutions for film use, licor solutions for western blotting, the fall of film use for western blots, video infographic, western blot imaging | Leave a comment

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

Posted on December 10, 2013 by licor-admin

As 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.

Not 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.

The Cost of Film Production May Give Us One Clue Why Film May Not Be Available for Western Blot Imaging in the Future?

Posted on November 26, 2013 by licor-admin

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:

(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.

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?.

Posted in C-DiGit System, Odyssey Imaging Systems, Western Blotting | Tagged chemiluminescent western blot imaging, chemiluminescent western blots, cost of film, cost of film raw materials, digital imaging for western blots, film production is expensive, imagej software alternative, li-cor western blotting, using film for western blot imaging, western blot imaging, x-ray film production is expensive | Leave a comment

Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals

Find Your Perfect Match with IRDye^® and VRDye™ Secondary Antibodies!

Straight from Cupid for specific bonding!

Happy Valentine’s Day from LI-COR!

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

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

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

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

Give the Gift of Quantitative Western Blots and Be the Hero in Your Lab this Holiday!

Video Infographic: The Fall of Film and Its Effect on Your Western Blots

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

The Cost of Film Production May Give Us One Clue Why Film May Not Be Available for Western Blot Imaging in the Future?

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Straight from Cupid for specific bonding!

Happy Valentine’s Day from LI-COR!

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