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.
The 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.
The 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.
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.