Want to Save at least 90 minutes the When You Do a Western Blot?

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!

Quick Western Kit Workflow

Scientists using Quantitative Western blotting and Odyssey® Infrared Imagers

The Odyssey Family of Imaging SystemsThere 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 publications database. Search by instrument, area of research, application and country to get a personalized list of publications related to your research.

Updated November 20, 2017.

Optimizing your Near-infrared Fluorescent In-Gel Westerns

In-Gel Western with Two-Color Detection A Powerful Technique for Large, Hard-to-transfer Proteins
The In-Gel Western 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

Love Traditional Western Blotting with Chemiluminescence Detection? Go Digital!

Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging SystemTired 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!

Schematic of Digital Chemiluminescent Imaging on the Odyssey Fc

The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. Learn about FieldBrite™ XT2 technology and the advantages it gives you with the Odyssey Fc.

The Odyssey Fc Imaging System can also be used for Coomassie-stained protein gel imaging and DNA gel imaging of either ethidium bromide-stained gels or SYTO® 60 stained gels.


Detect Difficult Proteins More Easily with Near-Infrared In-Gel Westerns

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 or the Odyssey Fc 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.

Comparing Odyssey Infrared Detection of In-Gel Westerns vs. Chemiluminescence Detection

Figure 1. Sensitivity of Odyssey infrared In-Gel Westerns is equal to or better than chemiluminescence. Beginning with 10 ng/lane (far left), two-fold serial dilutions of purified Transferrin were separated by electrophoresis on duplicate gels. In-Gel Westerns were detected with infrared fluorescence (top) and chemiluminescence on film (bottom). Odyssey detection outperformed chemiluminescence.


For more information, refer to Odyssey® In-Gel Western Detection Protocol and the In-Gel Western application pages.