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.
Traditional Western blotting is a labor-intensive process that includes gel electrophoresis, protein transfer to a blotting membrane, incubation with primary and secondary antibodies, and chemiluminescent or fluorescent detection of target proteins. (View a typical Western blotting workflow.) Day-to-day reproducibility is poor, because small variations in lysate preparation, gel loading, electrophoresis, transfer, and detection are unavoidable sources of technical variability.
We recently hosted a webinar called “Rethinking the Traditional Western Blot”, during which John Lyssand, PhD, from LI-COR Biosciences, discussed the In-Cell Western Assay and an example of its use in neuroscience research, in this case, Alzheimer’s Disease. The In-Cell Western Assay enables screening and analysis of many more samples in each experiment, eliminates error-prone protocol steps, and delivers higher reproducibility for biological and technical replicates.
If you didn’t have a chance to join us in September for “Rethinking the Traditional Western blot”, you can view this webinar online and on-demand. Check out the information on In-Cell Western assays on our website. You can also read Professor Dickey’s white paper as cited above that outlines how he and his group used higher throughput method to study Alzheimer’s Disease.
Have you ever wanted to try an in-cell ELISA but you just weren’t sure how to get started? With the new LI-COR®CellTag™ 700 Stain, a near-infrared fluorescent, non-specific cell stain that provides accurate normalization to cell number, you have a easier — and more affordable — way to try this powerful application. CellTag 700 Stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers (see Figure 1 below). CellTag 700 Stain is applied to the cells during incubation with IRDye® 800CW secondary antibody, and enables accurate measurement of target protein levels with much higher throughput than Western blotting.
Figure 1. Linear Relationship between Fluorescence and Cell Number. Two-fold serial dilutions of A431 and NIH/3T3 cells were plated in 96-well plate, then fixed, permeabilized, stained with CellTag 700 Stain, and detected with Odyssey Classic (Resolution: 169um; Quality: medium; Focus offset: 4.0mm; Intensity: 5). The Trim Signals were used to generate the graphs.
CellTag 700 Stain ICW Kits offer a convenient way to try cell-based In-Cell Western Assays. Each kit includes blocking buffer, IRDye® 800CW secondary antibody for detection of a specific protein target in the 800 nm channel, and CellTag 700 Stain to normalize well-to-well variations in cell number. This cost-effective normalization method makes quantification of the target protein more precise. Figure 2. In-Cell Western Assay with CellTag 700 Stain in EGF-stimulated A431 Cells. (Go to the CellTag 700 Stain Overview page for more details on this data).
Glycosylation is one of the most common and important events in post-translational modification. Over half of all proteins are believed to be glycosylated, and the resulting glycoconjugates play an important role in many biological processes. They have been connected to instances of cancer development, retrovirus infection, and other diseases. In an effort to understand these diseases, glycoprotein analysis has become a growing area of research. (See examples of typical glycan structures.)
Analysis of glycoproteins requires sensitive and quantitative applications. LI-COR offers a single, optimized solution using the Odyssey® Systems and IRDye® labeled conjugates to analyze glycoproteins. This solution provides sensitive and quantitative results using two-color near-infrared detection at 700 nm and 800 nm wavelengths. Operating at this wavelength produces lower background from biological materials, buffer components, and standard membranes used in Western blotting and lectin binding applications and, thus, superior data.
Outlined below are a variety of applications for several one-color, visible glycoprotein applications that have been adapted to near-infrared fluorescence detection on an Odyssey Imaging System:
Our IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:
IRDye 800CW (PN 926-32280)
IRDye 680RD (PN 926-68180) or
IRDye 680LT (PN 926-68080).
Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).
IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.
IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.
IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.
If so, do you know that the Odyssey® Fc Dual-Mode Imaging System now offers you the advantage of imaging DNA gels stained with ethidium bromide (EtBr), SYBR® Safe, and many other DNA stains using the 600 nm channel? How about that for multi-functionality?!
DNA or nucleic acid gel documentation is a common technique performed in the lab. Ethidium bromide is a common DNA stain. But, like many, if you are using SYTO® 60 as a near-infrared fluorescent DNA stain, then you can image your nucleic acid gel in the 700 nm channel of the Odyssey CLx, Odyssey Sa, OR Odyssey Fc. The detection sensitivity and lower limit of detection for SYTO 60 with any of these Odyssey imaging systems has proven to be better than with ethidium bromide detected with either a Polaroid camera or a CCD imaging system.
Don’t believe it? Check the data below, we think you may like what you see. In the figure below, DNA Gels imaged on the Odyssey Fc using Ethidium Bromide, SYBR Safe, and SYTO® 60. The Ethidium Bromide gel was also documented using Polaroid to show the comparison. All were imaged on the Odyssey Fc Imaging System.
So, you may ask, what’s so great about NewBlot Stripping Buffer?
Effectively removes antibodies, yet gentle enough to retain immobilized proteins
Strips blots at room temperature in 20 minutes or less without an unpleasant odor
Allows you to reuse the same blot up to 3X! (see the data below!)
Offers qualitative analysis after stripping
In the example below, beta tubulin and ERK2 were run on a gel and transferred to Immobilon®-FL PVDF membrane. They were probed with primary antibodies rabbit anti-beta-tubulin and mouse anti-ERK2 and then with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green), respectively. NewBlot PVDF Stripping Buffer was used to strip the blot, which was then reprobed with the fluorescent secondary antibodies. This was repeated 2 more times. As you can see from the series of images, there is very little apparent loss of signal in either channel in the third blot as compared to the original blot.
Note: On August 25, 2014, we launched two new Western blot stripping buffers: NewBlot™ IR Stripping Buffer for infrared Western blots on either PVDF OR nitrocellulose membranes; and, WesternSure® ECL Stripping Buffer for chemiluminescent Western blot stripping and reprobing. BOTH do not require hazardous shipping charges, unlike many other Western stripping buffers.
Figure 1. Linearity comparison of COX IV rabbit monoclonal primary antibody (P/N 926-42214) to β-Actin rabbit monoclonal (P/N 926-42210). Primary antibodies were compared by Western blot and detected with IRDye 800CW Goat anti-Rabbit (P/N 926-32211). The COX IV antibody can be used as a mitochondrial loading control and a loading control for normalizing low expressing target proteins. This COX IV primary antibody remains linear with increasing concentrations of lysate, making it ideal for normalization.
Also called “gel shift” or “gel retardation” assays, EMSA can be used to analyze sequence-specific recognition of nucleic acids by proteins.
Traditional, radioactive EMSA protocols can be easily adapted to near-infrared fluorescence EMSA detection by using IRDye end-labeled oligonucleotides and imaging with the Odyssey® CLx or Odyssey Classic Infrared Imaging System, providing a safe and sensitive alternative.