Would you like to save at least 90 minutes the next time you do a Western blot?

Posted on February 21, 2012 by admin

The Quick Western Kit — IRDye® 680RD (P/N 926-68100) provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step. This kit works with a variety of primary antibodies (see list) and has been shown to recognize primary antibodies to recombinant tagged proteins (i.e. 6X His, Myc, FLAG, etc.).

This reduces the overall time to complete a Western blot and provides the advantages of near infrared fluorescence detection.

If you do a lot of Western blots, this time saved could really add up fast. That’s a lot more experiments, reading, or sleep that you could catch up on! WOW!

Save time with the LI-COR Quick Western Kit

Posted in Applications, Odyssey CLx, Odyssey Family of Imaging Systems, Odyssey Fc, Odyssey Sa, Reagents, Western Blots | Tagged , , , , , , , | Leave a comment

Scientists using Quantitative Western blotting and Odyssey® Infrared Imagers

Posted on February 16, 2012 by admin

There are over 4000 peer-reviewed journal articles in which scientists have cited the use of LI-COR products and imaging systems for all types of research – from apoptosis, autophagy, and angiogenesis to RNAi studies, transcription factor assays, and virology – and many disciplines inbetween.

Here is a review of a recent publication in which quantitative Western blots were performed on the Odyssey Infrared Imaging System.

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington’s Disease Model

Joost Schulte, Katharine J. Sepp, Chaohong Wu, Pengyu Hong, J. Troy Littleton
Dept of Biology, Dept of Brain and Cognitive Sciences, The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA

PLoS ONE 6(8): e23841 (2011)

Huntington’s Disease (HD), a dominant neurodegenerative disorder, results from expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein. This study describes a high-content small molecule and RNAi screen for suppressors of neurotoxicity, using a Drosophila primary neural culture HD model. An mRFP-tagged pathogenic Huntingtin variant (Htt138Q) was expressed to induce disease phenotypes. Suppressors of neurotoxicity were identified, including lkb1 (an upstream kinase in the mTOR/insulin pathway) and four drugs (Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone). Quantitative Western blotting with the Odyssey Imager was used to monitor expression of Htt variants, and for in vivo validation of screen hits. The suppressors identified in this screen also restored viability in an in vivo Drosophila HD model.

If you would like to see more references, check out our quarterly Publication Lists (most recently: Autumn 2011 and Summer 2011) plus Publication Lists specific to cancer, RNAi, and In-Cell Western™ assays. All of these can be found in the Technical Resource Library (choose Odyssey CLx -> Publication Lists).

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Optimizing your Near-infrared Fluorescent In-Gel Western

Posted on February 14, 2012 by admin

A Powerful Technique for Large, Hard-to-transfer Proteins

The In-Gel detection protocol may require optimization for each target protein or gel type. Sensitivity of In-Gel Westerns may be lower than standard Western blots. (Transfer to a membrane concentrates the target protein, whereas in gels, protein is dispersed through the thickness of the gel.)

Use the following guidelines for optimization:

  • Optimization of primary and secondary antibody dilutions, as well as amounts of Tween® 20 in diluted antibodies, may be needed to achieve maximum signal and minimum background. Recommended Tween 20 concentration is 0.1%.
  • Try different buffers for dilution of the antibodies, including PBST alone, Odyssey Blocking Buffer, or milk. Changing the buffer solution may dramatically improve performance.
  • To avoid background issues, use high-quality ultrapure water. Rinsing previously used incubation boxes or trays with methanol can reduce background contamination on gels.
  • For experiments utilizing streptavidin labeled with IRDye® infrared dyes, add 0.01% SDS in addition to Tween 20 in the antibody diluents and wash buffer.

Here is a nice white paper reference on In-Gel Westerns:
In-gel Immunochemical Detection of Proteins that Transfer Poorly to Membranes
Michael J. Theisen and Mark L Chiu, Abbott Laboratories

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Love Traditional Western Blotting with Chemiluminescence Detection? Go Digital!

Posted on February 10, 2012 by admin

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 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).

Posted in Applications, Chemi Westerns, Chemiluminescent Western Blots, Odyssey Fc, chemiluminescence detection | Tagged , , , , , , , | Leave a comment

In-Gel Westerns on the Odyssey® CLx or Classic – Difficult Proteins Detected More Easily

Posted on February 7, 2012 by admin

Western blot detection of proteins requires separation of protein mixtures by electrophoresis, followed by transfer of the separated proteins to nitrocellulose or PVDF membranes for detection. In-Gel Western detection avoids transfer problems by directly detecting target proteins within the polyacrylamide gel matrix, using the Odyssey® CLx or Classic Infrared Imaging System.

The Odyssey® Infrared Imaging systems allow you to detect target proteins while still embedded in the gel – without transfer to a membrane – using near-infrared secondary antibodies, such as the LI-COR IRDye Conjugates. Using near-infrared fluorescence detection methods for In-Gel Westerns makes this a powerful technique. It saves time, reduces cost, and eliminates the variables introduced by the transfer step or subsequent blocking of the membrane. In-Gel Western detection can be performed with standard Odyssey reagents – no special kit is required.

Figure 1. Beginning with 10 ng/lane, two-fold serial dilutions of purified human Transferrin were subjected to electrophoresis followed by detection using an in-gel Western Protocol. Reprinted with permission from Urh, M et al. Poster presentation, Advances in Genome Biology and Technology Conference (2002).

For more information, refer to Odyssey® In-Gel Western Detection Protocol

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EMSA/Gel Shift Assays on your Odyssey® CLx or Classic Infrared Imager

Posted on January 26, 2012 by admin

Gel shift assays or electrophoretic mobility shift assays (EMSA) provide a simple method to study DNA:protein interactions. This assay is based on the principle that a DNA-protein complex will have different mobility during electrophoresis than non-bound DNA. These shifts can be visualized on a native acrylamide gel using labeled DNA to form the DNA-protein binding complex.

Do you know that you can easily adapt your existing mobility shift assay protocols by replacing the radiolabeled oligonucleotides with IRDye® end-labeled oligonucleotides?

And using the Odyssey CLx or Classic Infrared Imager, you can complete your EMSA in about 90 minutes – saving valuable research time.

Figure 1.
EMSA performed with IRDye 700 AP-1 oligos. Reprinted with permission from Electrophoretic Mobility Shift Assay (EMSA) Using IRDye Oligonucleotides.

And when you are ready to image, there is no need to remove the gel from the glass plates. This makes gel handling easier and allows running the gel further, if needed, after scanning is completed. Possible deformations or tearing of the gel while separating plates are also eliminated.

For more information, refer to our Technical Note on Infrared EMSA detection.

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NEW! Quick Western Kit reduces Western blotting Time by at least 90 min

Posted on January 20, 2012 by admin

Introducing the NEW! Quick Western Kit – IRDye® 680RD

The Quick Western Kit – IRDye® 680RD provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step.

Saves Time (View Workflow)

  • Does not require a separate primary antibody labeling step, saving time and antibody
  • Faster method of detection compared to the traditional 2-step method, which can take up to 4 hours
  • Reduces the total Western blot procedure by at least 90 min

The kit can be used to detect primary antibodies from a variety of hosts and has been shown to recognize primary antibodies to recombinant tagged proteins (i.e. 6X His, Myc, DDK, etc.)

IRDye 680RD Detection Reagent is known to have high specificity for IgG from:

  • Human
  • Mouse
  • Rabbit
  • Guinea Pig
  • Goat
  • Sheep
  • Pig
  • Cow
  • Cat
  • Dog
  • Donkey

The IRDye 680RD Detection Reagent does not work with:

  • Chicken

The Detection Reagent is known to have lower specificity for rat, horse, and hamster. The Detection Reagent has been specifically tested and qualified for Western blot applications. If additional specificity and/or affinity are required, please use IRDye conjugated secondary antibodies for detection.

Two-fold dilutions of crude lysate containing an overexpressed 6X-His tagged DNA polymerase was loaded in Lanes 3-7. A Histag molecular weight marker (Invitrogen) was loaded in Lanes 1 & 9. The nitrocellulose membrane was blocked with Odyssey Blocking Buffer and probed with anti-His Tag Rabbit Polyclonal Antibody (GenScript; 1:500) and IRDye 680RD Detection Reagent (1:1000) for 1 hour. The image was collected on the Odyssey CLx.



Ordering Information
Pack Insert

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New and Improved ELISA Detection on LI-COR® Infrared Imagers – sign up for our Webinar

Posted on January 17, 2012 by admin

Join for a free webinar on LI-COR’s new solution for your ELISA needs. This complimentary on-line training session is for researchers interested in performing ELISA applications on LI-COR® Infrared Imagers, using our new HRP and AP ELISA Substrates.

Training topics for this webinar include:

  • Introduction to new near-infrared ELISA HRP and AP Substrates
  • How these substrates easily fit in to your current protocol
  • Scanning and quantification of plate data using Odyssey software
  • Comparison to commercially-available colorimetric and chemiluminescent substrates
  • Hints and tips to help you perform ELISA successfully

Speaker: Teresa Urlacher
Date: Wednesday, January 25, 2012
Time: 8:30 AM (Check your local time.)
Time: 1:30 PM (Check your local time.)
Where: Online – login info sent upon registration
Length: 30 minutes, plus Q&A

Posted in Applications, ELISA, Odyssey CLx, Odyssey Sa, Reagents | Tagged , , , , , , , | Leave a comment

Advantages of using PSVue® 794 for Imaging Apoptosis

Posted on January 11, 2012 by admin

PSVue® 794 is a near-infrared fluorescent probe for detection of apoptotic and necrotic cells, bacteria, and other anionic membranes. The compound exhibits fluorescence excitation maximum at 794nm and emission maximum at 810 nm and through its zinc(II)-dipicolylamine (Zn-DPA) moiety, it has been found to bind strongly to negatively charged bacterial cell walls (e.g. S. aureus, E. coli) and necrotic regions present in various tumors (e.g. mammary, prostate, glioma) in vitro and in vivo. In particular, it has also been found to bind to the phosphatidylserine (PS) residues exposed on the cell surface of apoptotic cells, making it a more cost-effective alternative to fluorescently-labeled Annexin V in various cell death assays.


Figure 1. MPTP was used to induce cell death in mouse brains as a model for Parkinson’s Disease. C57BI/6 mice were treated with MPTP to selectively destroy dopaminergic neurons. Mice were then injected with PSVue dye or control dye and imaged on the Pearl® Imager 68 hrs post injection. A. control (i.e. non-targeting) dye; B. and C. PSVue dye; D. excised brains from the three animals.

Download a scientific poster presenting information on the use of PSvue 794 in studying Alzheimer’s Diesase, Parkinson’s Diesase, and contact dermatitis in mouse models.

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Chemiluminescent Western Blots – HRP-Conjugated Secondary Antibody Selection

Posted on January 6, 2012 by admin

So we’ve talked about primary antibodies (see our previous posts), and you have come to know the ones you are using very well. The next step in Western blotting detection (after washing the blot, of course) is to probe with the secondary antibody (also known as secondaries, secondary conjugates, antibody conjugates).

As you can see in the images below, the reactivity of secondary antibodies ranges widely between vendors, even within the same species and especially between host species. The ratio of HRP enzyme to antibody varies and may affect the detection of the target. Try secondary antibodies from several vendors to find the ones that give the most satisfying data.

GAM 2nd Ab Vendor A GAM 2nd Ab Vendor BGAM 2nd Ab Vendor C

Serial dilutions of mouse or rabbit IgG were spotted onto nitrocellulose (2500 pg to 0.3 pg) and probed with HRP-conjugated secondary antibodies from various vendors. Blots were detected with SuperSignal® West Dura chemi substrate (Thermo Scientific) and exposed to film for 15 seconds.

Another one of those Notes: When evaluating the performance of the primary and secondary antibodies, try different blocking buffers (yes, we had a post on that too – 8-Dec-11 – The Best Offense is a Good Blocker), as the choice of blocker can affect the antibodies’ performance.

For optimal results do not dilute the HRP-conjugated secondary antibodies with blocking buffer containing sodium azide as a preservative (e.g., Odyssey Blocking Buffer), as it will inhibit peroxidase activity and result in less light production.

Don’t want to use film?? (ugh! all that mess and expense!) Then, you might be interested in the Odyssey Fc Dual-Mode Imaging System.

For more optimizing tips for chemiluminescent Western blotting, see our Technical Note. And, of course, check in here again for more tips and hints on various applications that can be performed on the Odyssey and Pearl imagers.

Posted in Applications, Chemi Westerns, Chemiluminescent Western Blots, Odyssey Fc, Western Blots | Tagged , , , , , , , , , , , , | Leave a comment