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.
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.
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.
Have 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.
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. 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).