Monitor Protein Levels and Phosphorylation with Quantitative Multiplexed Western Blots

Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer’s disease human skin fibroblasts.

Mendonsa, G., et al. PLoS ONE 4(2): e4655. doi:10.1371/journal.pone.0004655

Aberrant signal transduction is associated with Alzheimer’s disease (AD). In skin fibroblasts of AD patients, exaggerated signal transduction occurs in response to bradykinin (BK), an inflammatory neuropeptide. BK-induced PKC signaling causes stimulation of tau phosphorylation on serine residues in AD fibroblasts, but not in normal skin fibroblasts. Quantitative Western blotting with multiplex fluorescent detection (Odyssey Imager; LI-COR Biosciences) was used to monitor protein levels and phosphorylation.

To explore the roles of inflammatory and oxidative stress in AD pathology, this study profiled the effects of these stresses on MAPK signaling cascades in human skin fibroblasts of familial AD patients. AD fibroblasts of different genetic origins express presenilin (PS-1 or PS-2) mutated at a variety of sites. These mutations caused diverse responses to stress induced by BK or H2O2, with unique profiles of stress-induced MAPK activation, caspase-3 cleavage, and survival pathway activation. These results indicate that AD research must consider a broad spectrum of inflammatory, oxidative, and other stress factors and intracellular signaling responses.
Reduced ERK activation in PS-1 (M146L) Alzheimer's disease fibroblasts stimulated with bradykinin.

Figure 1. Reduced ERK activation in PS-1 (M146L) Alzheimer’s disease fibroblasts stimulated with bradykinin (BK). These fibroblasts carry a mutation in presenilin-1 associated with aberrant signaling. Mutant and control human skin fibroblasts were treated with 250 nM BK and immunoblotted for active and total ERK. Odyssey Imager was used, and fold activation was quantified. Total ERK is shown in green, and phospho-ERK in red; overlapping signals (active ERK) are shown in yellow. ERK activation was greatly reduced in PS-1 (M146L) AD fibroblasts. Graphs show mean + S.E. *p < 0.05 and **p < 0.005; n = 4. doi:10.1371/journal.pone.0004655

In-Cell Western™ Assay Webinar – Applications Review

In-Cell Western Assays - Fluorescent ImmunoassaysFor those of you that like to watch videos and listen to information, here is a great webinar on In-Cell Western Assays.

In this In-Cell Western webinar, the basics of ICW assays are covered and these applications:

For more information on these plate-based fluorescent immunoassays, go to the In-Cell Western assay application page. There are also several sample protocols and information on how to set up, optimize, and analyze ICW assays.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea


Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

Cells in Suspension for Quantitative Cell Signaling Analysis using In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent Immunoassays

So I’ve talked about how to ensure that suspension cells attach to plates, when to know you have a monolayer, and why round bottom plates are the best when doing In-Cell Western Assays with suspension cells in the 23-May-12 blog post. On 29-May-12 post, the post discussed how to wash your microplates so that you don’t lose cells plus some troubleshooting tips. I hope you found both of those posts helpful.

Here is the last post in this series on using non-adherent cells for ICW assays. These are a few additional questions you may have about using suspension cells for this powerful immunofluorescent assay.

  1. What suspension cell lines have been tested for use in In-Cell Westerns?
    Suspension cell lines tested include Jurkat, K-562, and THP-1.
  2. What pathways have been tested?
    Pathways tested include ERK activation and apoptosis using cleaved caspase-3 as a marker (Figure 1). A sample protocol can be downloaded here.

Do you have other questions? Super! Please contact us and let us know how we can help you in your research. And stop by this blog again for more technical tips and troubleshooting hints on other applications.

Anisomycin induced Apoptosis in Jurkat Cells

Figure 1. Anisomycin-induced apoptosis in Jurkat cells. The image represents a 96-well two-color In-Cell Western assay with the 700 and 800 nm channels detecting TO-PRO®-3 DNA staining and cleaved caspase-3 (Asp175), respectively. The image was scanned using the Odyssey® Sa Infrared Imaging system with scan setting of 200 μm resolution, focus offset of 3.5, and intensity of 3.5 (700 channel) and 4 (800 channel). Background (B) wells were incubated with a secondary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the increase in caspase-3 cleavage in response to anisomycin treatment for 3 hours in Jurkat cells.

Additional resources:
In-Cell Western Assays: FAQs when Using Suspension Cells
Complete Sample Protocol for PMA-Induced ERK Activation in Suspension Cell Lines
LI-COR BIO Website

More Essential Success Tips for Performing In-Cell ELISAs Using Non-Adherent Cells

Round Bottom Plates for In-Cell Western Assays

I mentioned in my post on 23-May, that the next few entries would be on more hints and tips of how to use non-adherent cells for In-Cell Western Assays – so here goes!

During my washing steps, cells are coming off the plates.

  1. Are you using the recommended round bottom 96-well plate (BD Biosciences, P/N 353077)?
    1. If no, cells will more easily detach from the flat bottom plates than the round bottom
      plates. The multi-channel pipettors will generate enough pressure when expelling liquid from the pipet to cause cell detachment when using flat bottom plates. Cells will detach even when pipetting down the sides of the wells.
    2. If yes, make sure you pipet down the sides of the wells and not directly onto the cells. If this doesn’t help, you may need to change your multi-channel pipettor because different brands of pipettors have different amount of pressure required to expel the liquid from the pipet. The recommended multi-channel pipettor is the 12-channel Finnpipette
      (Thermo Electron Corp, P/N 4610050).
  2. Are you shaking or rotating the plates at a moderate to high speed?
    1. If yes, gentler shaking/rotating is needed to prevent cells from detaching. Cells will detach. Set shaking or rotating speed to very low speed.
    2. If no, are you dumping the solutions straight from the plates? Dumping causes cells to
      detach. Either aspirate very slowly or manually pipet using the sides of the wells.

Why can’t I use the flat bottom 96-well plates?

  • LI-COR® Biosciences recommends using the round bottom 96-well plates for the reasons mentioned above.

When I scan an empty round bottom 96-well plate, I get lots of background noise.

  • The round bottom plate shows some background autofluorescence. The background fluorescence is relatively small compared to signal (about 200-fold difference depending on the intensity of the signal) and can be subtracted from the signal. It is necessary to include background wells containing cells and only the secondary antibodies in order to
    completely subtract away the background noise originating from the plate as well as from the non-specific binding of the secondary antibodies.

Here is a technical note on more FAQs on using non-adherent cells for In-Cell Western Assays. Or you can just stay tuned to my next blog post!

To your Research Success!

Using Non-adherent Cells for Fluorescent Immunoassays – Tips for Successful In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent Immunoassays

You might be wondering if this powerful technique called In-Cell Western Assay can be used for your cell line because your cell line is non-adherent. Well, you are in luck! You CAN use suspension cells for ICW Assays – with some care and optimization.

Here are a few frequently asked questions. (see my next few blogs for more FAQs on using suspension cells for In-Cell Western Assays).

  1. How do you make non-adherent cells (suspension cells) attach to plates?

  2. A simple trick is to replace your complete media containing 10% serum (usually fetal bovine serum) with the same media minus the serum. Then allow the cells to sediment, forming a monolayer of cells within 10 minutes. Caution: Although cells appear attached to the plates, they are relatively loosely attached and therefore, extreme caution is required during solution-changing steps.

  3. How do I know that I have a monolayer?
    Method #1
  4. – Examine cells in the round bottom 96-well plates under a light microscope. The center of the wells should all have a small flat circular surface area where all the cells in that field are “in focus”. Moving the plane of focus, up or down, will cause cells to be “off focus”.
    Method #2 – Hold the round bottom 96-well plate under a light source. The monolayer should look opaque rather than transparent. Cells will not attach on top of the cell monolayer, so the opaqueness is due only to the monolayer.

  5. I cannot get a monolayer of cells. I get lots of spaces between cells. Is seeding 200,000 cells/well enough?
    Seeding 200,000 cells/well is more than enough to form a complete cell monolayer. It is necessary to allow the cells in serum-free media to sediment in the T75 flask (or other tissue culture plates) for approximately 30 minutes before counting cells using a hemacytometer. When cells in serum-free media are placed, for example, in a T7 tissue culture flask, a monolayer of cells will immediately begin to form on the bottom of the flask. This will dramatically decrease the number of cells in suspension that are available for plating.

    Note: Once a complete monolayer has formed on the plate, the rest of the cells will remain in suspension. Count these cells in suspension and the cells attached to the T75 flask can be discarded later.

Here is a complete sample protocol for PMA-induced ERK Activation in Suspension Cell Lines.

Check on the website pages on Tips for Using Cells in Suspension Cells for In-Cell ELISA Assays.

Fast, Cost-Effective Cell-Based Assays for Quantitative Cell Signaling Analysis

In-Cell Western Assay Kits for Quantitative Cell Signaling Analysis
The In-Cell Western™ Assay is an immunocytochemical assay that uses near-infrared fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Using the In-Cell Western Assay kits, the cost per well for secondary screening is reduced to a fraction of the cost of typical screening methods. Watch an introductory webinar to In-Cell Western Assays.

The CellTag™ 700 Stain ICW Kits provide antibodies, blocking buffer, and CellTag 700 Stain to normalize well-to-well variations in cell number for forty 96-well plates or ten 384-well plates. 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.
Phospho-ERK Analysis with Sapphire700/DRAQ5 for Normalization

Additional Resources:

Other In-Cell Western Assay Protocols are available on our Technical Resources Library. Visit our In-Cell Western Application Pages for more examples of how researchers have used these immunocytochemical assays.

Updated February 18, 2015.

Are You Studying Phosphorylation or Quantitative Cell Signaling Analysis? How About for IC50 Determinations?

In-Cell Western Assays - Fluorescent Immunoassays

In a previous post, 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.
Use of labeling for In-Cell Western Assay normalization.
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).

Dose titration of Wnt3a treatment of mouse L-cells.  An In-Cell Western Assay Application.
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!

Use Quantitative Cell Signaling to Study Apoptosis

AGAIN with the quantitative cell signaling! YES! because it is so versatile!! I am sure you will find that this will become a valuable technique to use in your research.

This quantitative immunofluorescent assay – the one that we call an In-Cell Western™ (ICW) 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.

Time Course of Caspase-3 Activation in SP2 Cells Performed using an In-Cell Western Assay

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).

Quantitative Cell Signaling Analysis – What’s all the Buzz About?

In-Cell Western Assays for Quantitative Cell Signaling Analysis

What’s all this BUZZZZ you are hearing about being able to quantitate cell signaling in plate-based assays? If you are at AACR in Chicago this week, stop by Booth 3800 (LI-COR® Biosciences) and we can tell you all about the In-Cell Western™ Assay – and how you can use this method to quantitate signaling, look at levels of protein phosphorylation, perform RNAi studies, monitor gene expression levels, conduct cell proliferation assays, and more. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imager (the Sa also has the option for automation and barcode reading). And, if you can’t make it to AACR, stay tuned here and I will be blogging about this topic over the next week or so.

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 (ICE Assay). 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 a brochure on near-infrared applications for the Odyssey Imaging Systems, 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!

Updated October 6, 2016.