Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

Example of Multiplex Western Blotting using the MPX Blotting SystemMultiplex Western blotting is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

  • Primary antibodies – to determine the primary antibody that has the right specificity and the right dilution for use
  • Antibody incubation times
  • Blocking conditions – which blocking buffer will give you the optimum results
  • Secondary antibodies – what dilutions is best to use without getting a lot of non-specific binding?
  • Or just about anything else you need to optimize!

Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

Rethinking the Traditional Western Blot

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.

Snapshot of In-Cell Western Assay MethodThe In-Cell Western™ (ICW) Assay, a quantitative immunofluorescent method, is an alternative to traditional Western blots that increases both reproducibility and sample throughput. (View a typical ICW workflow.)

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.

ICW Use: Tau Protein Accumulation and InhibitionThe data presented demonstrated how ICW assays were used in Alzheimer’s Disease research to screen HSP90 inhibitors for their effectiveness in reducing tau activity levels. Dr Lyssand discussed how and why the In-Cell Western Assay is superior to traditional methods for screening of cell samples.

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.

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. 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 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.


Check out some of our Publications Lists for:

Create a Complete Molecular Imaging Workstation

pearltrilogybuildsystemCombining the Odyssey® CLx Infrared Imaging System with the Pearl® Small Animal Imaging System creates a versatile molecular imaging workstation for in vivo and in vitro imaging.

BrightSite™ Optical Imaging Agents or probes developed using IRDye® infrared dyes can be used for in vitro, in vivo, and tissue imaging. This technology offers researchers the ability to take research from the cell to the animal, all within one lab.

Odyssey CLx Infrared Imaging System Capabilities:

  • Cell-based assays (binding capacity, specificity, competition, etc.) for optical agent development
  • Histology and whole organ imaging for studying clearance and specificity
  • Simultaneous two-color detection for two targets or one target with sample normalization

Pearl Small Animal Imaging Capabilities:

Validation Workflow and Molecular Imaging WorkstationFigure 1. Validation and Use of an IRDye Fluorescent Probe. After probe labeling, in vitro cellular assays and microscopy are used to confirm specificity. The desired target is then imaged in animals. Excised organs and tissues> can be examined for more detailed localization of the probe. Animal image captured with Pearl Imaging System. A more comprehensive discussion of approaches for the development of fluorescent contrast agents has also been published. Reference: Kovar, et al. Anal Biochem 367(2007) 1-12.

Molecular imaging – achieved with near-infrared fluorescent technology from LI-COR!

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 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.