New Cell Stain Increases Ease of Use for In-Cell Western™ Normalization

CellTag 700 Stain ICW Kits for Quantitative Cell Signaling AnalysisHave 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. The 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.

CellTag 700 Stain - Linear Relationship between Fluorescence and Cell Number.

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 Normalization 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.
In-Cell Western Normalization with CellTag 700 Stain in EGF-stimulated A431 Cells.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).

Try one of our new In-Cell Western Assay Kits with CellTag 700 Stain today and find out just how easy it is to perform fast, cost-effective cell-based Western assays.

Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.

Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.

Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.

Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.

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Analyze Glycoproteins with Sensitive, Quantitative Infrared Fluorescent Techniques

O-Linked Glycan StructureGlycosylation 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. 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:

Read Glycoprotein Detection with the Odyssey Infrared Imaging System for more indepth information on using your Odyssey Infrared Imaging System for glycobiology research.

Easy-to-Use Image Studio v3.1 Software Now Available for all Odyssey® and Pearl® Imaging Systems

Image Studio Software
from LI-COR® is available for Mac® and Windows® computers. This intuitive software allows researchers to:

  • Create individual work spaces
  • Import tif, jpg, or png images from Odyssey, Pearl®, or other imaging systems
  • Image and export data
  • Customize lab notebooks

Upgrade your current Odyssey or Pearl Imager software and all the benefits of this new, improved Image Studio Software.

If you would like to get an idea of how easy Image Studio Software is to use, try our free version – Image Studio Lite. Image Studio Lite allows you to analyze Western blot images but does not operate your LI-COR instrument to acquire images. Try it FREE – download now!