• Detect and quantify target proteins localized to the cell surface
• Quantify ligand binding to cell surface receptors
• Monitor receptor internalization and recycling by detecting loss and re-appearance at the cell surface
• Perform and detect cell surface biotinylation assays
• Evaluate the effects of mutations, drugs, and other treatments on protein trafficking
• Analyze many samples quickly and quantitatively
• Avoid use of radioactivity

Figure 1. Cannabinoid receptor 1 (CB1) is internalized after exposure to a specific agonist (Win-2), but the effect is blocked by the antagonist SR1. A) Intensity levels were greatly reduced in wells treated with 1μM 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. LI-COR Biosciences application note (2004).

Visit our On-Cell Western Assay Application page for more information.
We have a sample protocol of On-Cell Western Assay for Targeted Near-infrared-labeled optical imaging agent development.
We also have a list of additional references on the uses of On-Cell Western Assays.

Image Studio is an extremely simple and easy-to-use imaging software. It is compatible with the Odyssey^® CLx, Odyssey Classic, and Odyssey Fc Imaging Systems.

• Easy to use – training for new users is fast and simple
• Intuitive, application-driven ribbon interface
• Supports nine different types of analyses, including Western blots, DNA gel documentation, and small animal imaging

NOW! Compatible with Mac and PC computers!

Watch a video on the Features and Benefits of Image Studio Software.

For more information on Image Studio Software for Mac and PC, visit our website.

The In-Cell Western™ Assay Kits provide antibodies, blocking buffer and stains for forty 96-well plates or ten 384-well plates. Sapphire700™ and DRAQ5™ protein stains are included to normalize for well-to-well differences in cell number. Using protein stains reduces the cost per assay compared to performing the assay using two secondary antibodies. Any potential interference caused by using two antibodies is also eliminated.










Additional Resources:
In-Cell Western Assay Protocol: Complete Apoptosis Assay Example Detailing the Seeding, Induction, and Detection of the HeLa Cellular Response to Anisomycin Treatment
Odyssey Publications List: In-Cell Western Assays, Vol 2, Winter 2012

Other In-Cell Western Assay Protocols are available on our Technical Resources Library.

LI-COR^® has just rolled out a new way that the recently-released Quick Western Kit – IRDye^® 680RD (see my blog post from 21-Feb-12) can be used in your research..

Not only can the Quick Western Kit reduce Western blotting time by 90 min, the kit ALSO serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot. Seriously, how cool is that!!??!!

Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample ; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.

For more information, visit our website. Here’s the pack insert. To order this product (online ordering available for US only), go to our ecommerce site.

In my post on April 5, I talked about how In-Cell Western™ assays could be used when studying apoptosis. So, you may be asking yourself, for what other applications can quantitative cell signaling analysis be used? GREAT QUESTION!!

Well, in-Cell ELISAs (as these immunofluorescent assays are also called) have been used successfully in studying protein phosphorylation. Whether you are looking at the effects of drug compounds on signaling pathways, or the timing/kinetics of signal transduction, or trying to determine the IC50 of compounds, In-Cell Western assays are a valuable tool.

Here are two examples of data from IC50 and EC50 determination experiments.

Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS-ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. For details, see Hoffman, GR et al. Assay Drug Dev Tech 8(2):186-99 (2010).

Figure 2. Dose titration of Wnt3a treatment of mouse L-cells. Half-maximal activation (EC50) of cellular beta-catenin levels occurs at 33 ng/ml ligand. Hannoush, RN. PLoS One. 3(10):e3498 (2008). Creative Commons license 2.5.

To help you get started in designing your experiment, here is a complete sample protocol for measuring IC50 of the inhibitor PD168393 in A431 cells responding to epidermal growth factor (EGF).

Check here for future blog posts on other applications of quantitative cell signaling analysis!

1. Plates: For most adherent cells that stick to wells tightly (e.g. A431, HeLa, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, such as Nunc P/N 167008. For adherent cells that could detach from wells during In-Cell Western assay wash steps (e.g. NIH3T3), we recommend Poly-D-lysine coated 96-well microplates.
2. Cell seeding density: Typically, 15,000 to 40,000 cells are seeded per well. Two to three days are usually required for cells to reach the appropriate confluency, depending on growth rate. Seeding with low cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.
3. Confluence: To obtain maximal fluorescent signals, complete or near complete confluency is recommended for cells that stick to wells tightly. For cells that adhere loosely to wells, such as NIH3T3, 70% confluency should be used. Please note that cell type and experimental conditions may affect the acceptable level of growth confluency.

The example below illustrates the importance of cell seeding density for A431 cells. As shown in the corresponding graph, cell growth is greatly inhibited when there are too few neighboring cells.

This quantitative immunofluorescent assay – the one that we call an In-Cell Western™ Assay – can be used to study a variety of mechanisms. Here is an example of an ICW used to study apoptosis.

As you may already know, there are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Under most circumstances, activation of either pathway leads to proteolytic cleavage and activation of caspases, a family of cysteine proteases that act as common death effector molecules. The In-Cell Western Assay is a very helpful research tool for scientists who are quantifying cell signaling.

Figure 1. Time course of caspase-3 activation in S2 cells. (A-C) In-Cell Western analysis of S2 cells treated with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A; green) and f-actin (B; red, stained with near-infrared fluorescent phalloidin). Panel C shows merged pseudocolor images. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was designated as 1, and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active caspase-3 over time. Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008).

Here is our complete apoptosis assay example protocol of the HeLa cellular response to anisomycin treatment (detailing the seeding, induction, and detection).

Okay, let’s start at the beginning. So what – exactly – is an In-Cell Western Assay? Well, some call it a cytoblot. To others, it’s a cell-based ELISA or an In-Cell ELISA. To LI-COR, it’s a In-Cell Western Assay (we call it an ICW, for short) and is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format). It combines the specificity of Western blotting with the reproducibility and throughput of ELISA.

In a nutshell, the basic steps are:

• Culture cells in microplates
• Treat cells
• Fix and permeabilize
• Stain with primary antibodies – 1 or 2 protein targets per well
• Stain with IRDye secondary antibody conjugates
• Image microplate and quantify fluorescent signals from cell populations in each well
• Quantify relative protein levels
• Normalize to correct for well-to-well variation

That doesn’t sound too difficult, right? Of course, just like any scientific technique, there are things to keep in mind to make sure your experiment gives the best, clearest, most accurate and reproducible results it can. In the next posts, I’ll share some of the technical tips to keep in mind – plus examples of how your research colleagues have used In-Cell ELISAs in their published papers.

In the meantime, here is the ICW Brochure, which includes a little more info on the technique and some examples with data. We also have a video introduction to In-Cell Western Assays – for those that like the movies!

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.

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.

Here are some examples of what you can do with near-infrared dyes and reagents:

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.