LI-COR is expanding its portfolio of reagents by offering VRDye™ 490, VRDye 549, and IRDye® 650 dye-labeled secondary antibodies and protein labeling kits. These new secondaries can be used for for a variety of applications, including immunofluorescence microscopy and flow cytometry. Just like our IRDye dye-labeled secondary antibodies, these new visible fluorescence antibodies are highly cross-adsorbed. The dyes are conjugated to the same antibodies as the existing IRDye secondary antibodies, which are used for Western blotting and In-Cell Western™ Assay applications. This gives researchers the ability to correlate microscopy and flow data with Western blot and cell-based assay data. The VRDye secondary antibodies are suitable for multiplex experiments when combined with other secondary antibodies labeled with proper fluorescent dyes and using instrumentation with appropriate excitation and detection capabilities.
Figure 1. Immunofluorescence staining of tubulin protein in HeLa cells. Cells were cultured on cover slips. After fixation and permeabilization, cells were incubated with rabbit anti-tubulin mAb (CST), followed by VRDye™ 490 Goat anti-Rabbit IgG (LI-COR P/N 926-49020). Nuclei were stained with DAPI. Image acquired with Olympus IX81 microscope.
Figure 2. Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides. F98-EGFR tumors were snap-frozen in O.C.T. ™ compound and sectioned at 4-µm thickness. After fixation and permeabilization, cells were incubated with rabbit anti-EGFR mAb (CST), followed by detection with VRDye™ 549 Goat anti-Rabbit IgG (LI-COR P/N 926-54020). DAPI was used to stain the nuclei. Image acquired on Olympus IX81 microscope.
In addition, many researchers use labeled primary antibodies for flow cytometry. LI-COR now offers visible fluorescent dye protein labeling kits that are ideal for customers who need to label custom monoclonal antibodies for this application.
Visit our website for more information on these new visible fluorescence antibodies and protein labeling kits or to order them for your research.
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).