Do you know that LI-COR® near-infrared dyes and reagents can be used to perform microscopy? Absolutely! While we do not sell microscopes or offer microscopic equipment, we have evaluated the near-infrared detection capabilities of microscopes from several manufacturers, particularly in the ~800 nm wavelength region.
Here are some examples of what you can do with near-infrared dyes and reagents:
Figure 1. Deconvolved image of IRDye 800CW EGF binding to an A431 cell. Red represents IRDye 800CW EGF (P/N 926-08446); green represents Sytox Green nuclear stain (Invitrogen). Image captured using a Zeiss AxioImager microscope outfitted with xenon light source, IRDye 800CW custom filter set from Chroma Technology (EX: HQ760/40x, DC: 790DCXR, EM: HQ830/50m), and CCD camera with extended spectral range.
Figure 2. Staining of duplicated centrosomes. Condensed chromosomes are stained with DAPI (blue). The two centrosomes (red dots) are stained with a primary antibody against pericentrin (a centrosomal component) and IRDye 800 secondary antibody. Image captured with a Leica DM RXA epifluorescent deconvolution microscope. Outfitted with xenon light source, IRDye 800 filter set from Chroma Technology (EX: HQ740/35x, DC: 770DCXR, EM: HQ780LP), and Cooke Sensicam CCD camera without extended spectral range (quantum efficiency for IRDye 800 emission ~5-10%). Images courtesy of Mark Winey and Harold Fisk, Dept. of Molecular, Cellular, and Developmental Biology, Univ of Colorado Boulder.
Learn more about what you can do with near-infrared fluorescence detection when performing microscopy.
Are you studying receptor binding or cell surface protein expression? The On-Cell Western Assay is a great way to monitor cell surface proteins – quantitatively! – with near-infrared fluorescent detection. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imaging System.
With On-Cell Western assays, unpermeabilized cells are stained with antibodies against extracellular protein domains, so only cell surface antigens are detected. On-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear and black-sided plates with clear bottoms. Do not use plates with white wells, since the autofluorescence from the white surface will create significant noise.
Here is some data from a white paper by J. W. Miller:
Figure 1. Antibodies used were targeted against specific extracellular or intracellular domains of the CB1 (cannabinoid 1) receptor. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging.
Figure 2. Intensity levels were greatly reduced in wells treated with 1 μM of the CB1-specific agonist, Win-2. Cells treated with Win-2 and the specific CB1 antagonist, SR1 displayed no reduction of signal with the treatment. B) Graph displaying results of three independent experiments done in quadruplicate. Reprinted with permission from Miller, J.W. Tracking G protein-coupled receptor trafficking using Odyssey imaging.
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!
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 gel 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.
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.
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:
- Guinea Pig
The IRDye 680RD Detection Reagent does not work with:
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 (PBS) 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.
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 Disease, Parkinson’s Diesase, and contact dermatitis in mouse models.
For more near-infrared fluorescent probes, learn about BrightSite™ Small Animal Imaging Agents and CellVue® Burgundy Fluorescent and CellVue NIR Fluorescent Cell Labeling Kits.
We’ve discussed some hints on how there can be considerable difference in primary antibodies – so “Know thy Primary Antibody.“
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.
What questions do you have?
Optimizing Chemiluminescent Western blots Technical Note might be a good place to start to get some of those questions answered. And remember you save time and money by going digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!
LI-COR interviewed Dr. Go van Dam, a surgeon specializing in oncology at the Groningen University Medical Center in the Netherlands.
A key focus of van Dam’s research is to explore new tools such as targeted fluorescent imaging probes that will help address the challenges facing oncology surgeons. He discusses his research using near-infrared fluorescent imaging during surgery to improve cancer patient outcomes. Watch an interview with Dr. van Dam.
Vasilis Ntziachristos, PhD, Technische Universität München, Germany and Gooitzen M. van Dam, MD, PhD, University Medical Center Groningen, Netherlands presented “Shining New Light on Clinical Fluorescence Imaging” at World Molecular Congress in San Diego, CA in September 2011.