IRDye BoneTag optical imaging agents are tetracycline derivatives that incorporate into mineralizing bone. Structural imaging of bone can be used to more precisely localize an area of disease. A second disease-specific targeting agent with a spectrally-distinct fluorescent label can be used to localize and track disease (such as a tumor) in the same animal. When the two images are overlaid, bone structure is displayed in one color and the other target appears in a different color.
IRDye 680RD BoneTag and IRDye 800CW BoneTag are part of the ready-to-use BrightSite™ optical agents family and make it easy to begin animal studies immediately. These bright fluorescent agents are labeled with IRDye fluorophores for NIR fluorescence optical imaging, and they target a variety of disease characteristics. Simply administer the agent, then image with any small animal imaging equipment with appropriate 680 nm or 800 nm filter sets. No engineered cells or animals are needed.
Figure 1. IRDye 680 BoneTag agent for imaging of bone structure and remodeling. Tetracycline-derived probe reveals skeletal structure, and signal is stable for weeks. Dorsal view of mouse imaged with IRDye 680 BoneTag. Image acquired with Pearl® Small Animal Imaging System.
We’ll be at AACR in Chicago, April 1 – 4, Booth 3800. Stop by and talk to us about how you can start your small animal in vivo imaging experiments today.
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.
Okay, so you’re doing an in-gel western because you have a hard-to-transfer target (say, a glycoprotein). And you are using near-infrared fluorescence detection because it gets rid of inconsistencies due to transfer (and with an Odyssey it’s really fast and efficient to image and analyze!)
You read the In-Gel Western troubleshooting blog from March 6, 2012, but right now, what you’re seeing, or, um, well, NOT seeing is a signal. Rats! Where IS it?
Well, here are some possible causes with ways to solve or prevent this from happening:
- Not enough antibody.
- — Increase amount of primary and/or secondary antibody. Extend primary antibody incubation to overnight at 4°C to increase signal.
- — Remember that In-Gel detection is not as sensitive as blot detection; adjust sample loading and antibody concentrations accordingly.
- Antibody dilution buffer is not optimal for your primary antibody.
- — Try a different dilution buffer; this can significantly affect performance of some primary antibodies.
- — Suggested buffers include 3-5% BSA, Odyssey Blocking Buffer (PBS), or Odyssey Blocking Buffer (TBS), and PBS or TBS (all with 0.1% Tween® 20). Other blockers (milk, casein, commercial blockers) and Tween 20 concentrations can also be tested.
- Gel type is not optimal.
- — Amresco NEXT gels or NuPAGE® Bis-Tris pre-cast gels are recommended for In-Gel detection. Other commercial gel sources and homemade gels can be used, but may show reduced sensitivity and require further optimization.
- Antibody did not penetrate gel sufficiently or evenly.
- — Acrylamide percentage was too high. Try a lower percentage or a gradient gel.
- — Increase volume for antibody incubations so that gel is completely immersed in antibody solution.
- — Make sure gel is adequately fixed. Some monoclonal antibodies may be sensitive to residual acid in the gel; in this situation, eliminate acetic acid from the fix or extend the water wash step.
- Gel was left in isopropanol/acetic acid too long.
- — This may cause protein to be lost from the gel. Fix for 15 minutes only.
Whew! Well, hopefully by using one of these tips, you are NOW seeing a signal from your protein. Stay tuned for more troubleshooting tips for near-infrared fluorescent In-Gel Westerns in future blogs!
In-Gel detection of Cytochrome P450 3A4 (CYP3A4). Fixed gel was probed with anti-CYP3A4 primary antibody and IRDye® 800 secondary antibody. The limit of detection is approximately 3 ng. Reprinted with permission from Theisen, M. J. and Chiu, M. L. LI-COR Biosciences (2004)
So, you’ve selected your primary antibodies with care (see Know Thy Primary Antibody), and you’re using a great HRP-conjugated secondary antibodies. NOW – what about the chemiluminescent substrate?
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:
Updated February 18, 2015.
So, you are doing an in-gel western because you have a difficult-to-transfer protein. Good for you!! But, you are seeing high background – and now you need some help to optimize your application.
What causes high background on In-Gel Westerns? Here are some possible causes with suggestions on how to solve or prevent the high background from occurring.
- Stacking gel is still present.
- – Cut the stacking gel away after electrophoresis.
- Too much antibody.
- – Reduce concentration of secondary antibody.
- Uneven gel background may result from insufficient solution volumes for incubations.
- – Use enough solution at each step (fixation, washes, and antibody incubations) to completely immerse the gel.
- Pressing or squeezing gel during fixation and staining can cause splotchy background.
- – Handle the gel gently, with gloved hands, and by the edges whenever possible.
- Gel was not thoroughly washed.
- – Use plenty of wash buffers to allow gel to move freely. Do not allow the gel to stick to bottom of container.
- – Extend wash times or increase number of washes. Background may decrease if the gel is allowed to soak in PBS overnight at room temperature (protect from light).
- Contaminated scanning surface.
- – Before each use, apply methanol or ethanol followed by ultrapure water and wipe with lint-free tissues to remove residual dye. Remove any visible smears with isopropanol. Use canned air to remove any lint or dust.
Hopefully, after using some of these troubleshooting tips, you will get a nice gel image like this one:
In-gel detection of Cytochrome P450 3A4 (CYP3A4) Fixed gel was probed with anti-CYP3A4 primary antibody and IRDye® 800 secondary antibody. The limit of detection is approximately 3 ng. Reprinted with permission from Theisen, MJ and Chiu, ML. In-gel immunochemical detection of proteins that transfer poorly to membranes. LI-COR Biosciences (2004).