In this series of posts, you will discover how easy it is to use Image Studio™ Lite. This free Western blot analysis software from LI-COR® allows you to easily create your own work area and then import images from numerous sources. So, if you have an old film scan, or an image from another chemiluminescent Western blot imaging system, try using this free Western blot analysis software. In subsequent posts, we will talk about the other functionalities of Image Studio Lite.
This second video walks through the steps needed to import an image into Image Studio Lite. Image Studio Lite analyzes images in the tif, png, or jpeg format as well as images acquired with past versions of the Odyssey® or Pearl® imaging systems software.
WesternSure chemiluminescent Western blotting reagents offer the best performance available when compared to other competitive products on the market. WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots.
WesternSure HRP-conjugated secondary antibodies (Goat anti-Mouse and Goat anti-Rabbit) are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure PREMIUM chemiluminescent substrates.
Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?
FINALLY, image at your convenience. Keep your C-DiGit Blot Scanner on your lab bench, at your desk, or anywhere you choose (look left and see just how small and portable the C-DiGit Western Blot Scanner really is!). It can truly be YOUR personal chemiluminescent Western imager!
The C-DiGit Chemiluminescent Western Blot Scanner maintains the simplicity of film exposures without the mess of the darkroom. You perform all of the same steps, without buying film and spending time in the darkroom. The C-DiGit Scanner gives you a complete digital replacement for film – keeping the advantages of film and eliminating many of the drawbacks – saving you time AND money!
Watch this short video and then visit our website to get your very own C-DiGit Blot Scanner!
Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which Western blot detection method should you choose? Find out how the three common Western blot detection methods compare to each other in terms of time, sensitivity, and other important factors. The, choose what works best for your research.
Fluorescent detection: Fluorescent detection uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.
Enzymatic detection: Chemiluminescent and colorimetric methods use secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP). Signal-generating substrates are used.
Fluorescent detection uses NIR fluorescent dyes to generate a signal.
• Secondary antibodies are labeled with dyes such as IRDye 800CW or IRDye 680RD
• Digital imaging reveals target protein signals with high sensitivity
• Quantitative (signal is proportional to the amount of target protein present)
• Stable fluorescent signals are stable
• Multiplex detection of multiple protein targets without stripping and re-probing Chemiluminescent detection uses the horseradish peroxidase (HRP) enzyme and a luminescent substrate.
• Enzymatic reaction produces light that is detected by film exposure, or digital imaging with CCD camera
• Multiple exposures typically required to capture optimal signals and avoid signal saturation
• Very sensitive
• Cannot be multiplexed
• May not be quantitative
Colorimetric detection uses the alkaline phosphatase enzyme.
• Enzyme converts a soluble chromogenic substrate to a colored, insoluble product that precipitates onto the membrane and produces colored bands
• Development of the blot is stopped by washing away the soluble substrate
• Simple and cost-effective
• Limited sensitivity
Comparison of Chemiluminescence and Infrared Fluorescence Detection for Western Blotting
What’s all this BUZZZZ you are hearing about being able to quantitate cell signaling in plate-based assays? If you are at AACR in Chicago this week, stop by Booth 3800 (LI-COR® Biosciences) and we can tell you all about the In-Cell Western™ Assay – and how you can use this method to quantitate signaling, look at levels of protein phosphorylation, perform RNAi studies, monitor gene expression levels, conduct cell proliferation assays, and more. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imager (the Sa also has the option for automation and barcode reading). And, if you can’t make it to AACR, stay tuned here and I will be blogging about this topic over the next week or so.
Okay, let’s start at the beginning. So what – exactly – is an In-Cell Western Assay? Well, some call it a cytoblot. To others, it’s a cell-based ELISA or an In-Cell ELISA (ICE Assay). To LI-COR, it’s a In-Cell Western Assay (we call it an ICW, for short) and is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format). It combines the specificity of Western blotting with the reproducibility and throughput of ELISA.
Stain with primary antibodies – 1 or 2 protein targets per well
Stain with IRDye secondary antibody conjugates
Image microplate and quantify fluorescent signals from cell populations in each well
Quantify relative protein levels
Normalize to correct for well-to-well variation
That doesn’t sound too difficult, right? Of course, just like any scientific technique, there are things to keep in mind to make sure your experiment gives the best, clearest, most accurate and reproducible results it can. In the next posts, I’ll share some of the technical tips to keep in mind – plus examples of how your research colleagues have used In-Cell ELISAs in their published papers.
Yes, I know, there are tons of different substrates and vendors out there – all claiming to be the best, right? So, how do you choose the right one for your chemiluminescence Western blot?
One thing to keep in mind is that in this wide variety of chemiluminescent substrates for HRP detection, there are some that are better suited for digital Western imaging than others. In general, choose a substrate with a faster rate of reaction for use with the Odyssey Fc Dual-Mode Chemiluminescent and IR Fluorescent Imaging System or other digital chemiluminescent imaging systems.
Some substrates that are designed for optimal performance on film may not be suitable for detection on a CCD-based imaging system. Try different substrates to find the one that gives the most desirable image. As you can see from the images below, the substrate you pick DOES make a difference! So choose carefully!
In the three images below, two-fold serial dilutions of HRP-conjugated secondary antibody (1 ng-1.25 fg) were spotted onto nitrocellulose using a slot blot apparatus. Blots were detected with various chemiluminescent substrates.
Here are 2 documents with more troubleshooting information:
Tired of the darkroom being down or the expense of the development chemicals? Oh, AND all that film you go through because you have to do multiple exposures to get the image just right? Go DIGITAL with the Odyssey® Fc Dual-Mode Imaging System.
Dual-mode? Yes! The Odyssey Fc provides the ability to streamline your chemiluminescent Western blot imaging – no film, no darkroom, just clear fast blot images.
PLUS this digital imaging system includes two near-infrared fluorescent channels for sensitive, quantitative infrared Western blot imaging. So use ECL™ or whatever chemiluminescent substrate you usually use but eliminate film (more on best substrates to use in a later post). See how much money you will save going digital!
In addition, we’ve received some questions that are frequently asked – hence called frequently asked questions or FAQs – about primary and secondary antibodies when doing chemiluminescent Western blots. So here they are. I am sure there are more. . .so send them our way by commenting on this post!
Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.
Does it matter where I purchased the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. As well, the ratio of HRP enzyme to antibody varies, and may affect the detection of the target. If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.
Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.
Okay, so you’ve done your experiments, run your sample on a gel, and transferred the proteins to a membrane. Now, you need to see if you can detect the protein, what happened to it, how much is there, etc.
After you block (remember we talked about how important the right blocker is), you will probe with a primary antibody (that is, an antibody produced to detect a specific antigen) to see your molecule of interest. Now, primary antibodies can be produced in a wide variety of species such as mouse, rabbit, goat, chicken, rat, guinea pig, human, etc. There are lots of suppliers of antibodies out there, so it is important to realize primary antibodies for the same antigen can perform very differently. It may be necessary to test multiple primary antibodies for the best performance in your Western blot system.
In the images below, you can see how different primary antibodies to the same target may react. Serial dilutions of NIH/3T3 lysate were probed with Akt monoclonal primary antibodies from three different vendors. All blots were blocked with 5% skim milk and detected with HRP-conjugated Goat Anti-Mouse and SuperSignal® West Dura chemiluminescent substrate. Western blots were imaged on the Odyssey Fc Chemi channel for 2 minutes, shown with normalized image display settings. You can see that the primary antibodies varied quite a bit. The number and intensity of bands you can detect and the amount of non-specific binding that occurs are definitely different for each one.
So, take a cue from ancient Greece and get to know your Primary Antibody by doing some testing and optimization.
Okay, it’s football season, and I thought the analogy fit. 🙂 Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.
Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.
One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.
Why?, you ask.
Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.
Nota bene:Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.
On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.