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.

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.

Receptor-based Binding and Competition Assays with Infrared Fluorescence

What is an On-Cell Western? It’s a cell-based assay that enables quantitative monitoring of cell surface protein expression. The On-Cell Western assay offers the ability to:

  • 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

On-Cell Western Assay Example with Quantification

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 On-Cell Westerns for more information. We have a sample protocol of On-Cell Western Assay for Targeted Near-infrared-labeled optical imaging agent development. For other scientific publications in which On-Cell Westerns were used, visit our On-Cell Westerns Publications page.

Updated February 18, 2015.