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 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.

Normalize Protein Concentrations with Nucleic Acid Stains in In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent ImmunoassaysWhen performing the In-Cell Western assay, one fluorescence channel is often used for normalization. This allows quantification data for the protein of interest to be corrected for well-to-well variation in cell number, thereby increasing the overall accuracy of the assay. In many cases, a second protein is chosen for normalization.

LI-COR has a protocol for Nucleic Acid Stains Used as Normalizing Agents in ICW Procedures that describes the use of a DNA stain in the 700 nm channel as an alternative for normalization. Choosing to normalize with a DNA stain eliminates the need to identify and optimize an additional primary antibody for the assay, and avoids any issues that may arise if levels of the normalization protein are affected by the cell treatments used.
HeLa Cell Dilution Series to Measure Linearity of Various DNA Stains

The linearity of the DNA stains was evaluated with respect to cell number. HeLa cells were serial diluted in a 96-well plate at a starting concentration of 1.5 x 105 cells per well. Odyssey Blocking Buffer (PBS) was used to block for approximately 2 h before staining. DNA stains were diluted with fresh Odyssey Blocking Buffer at the recommended levels listed above. Blocking buffer was removed from the plate and blocking buffer plus DNA stain was added (50 μl per well). The plate was allowed to incubate at room temperature on a rotary shaker for 1 hour followed by 4X washes in 1X PBS + 0.1% Tween®-20. Care should be taken to protect the plate from light to insure the highest sensitivity of the stain. The linearity for SYTO® 60 and TO-PRO®-3 were very good with R2-values of 0.99 and 0.98, respectively (figure above).

For more information on In-Cell Western Assays, visit our website.

Request a LI-COR Catalog and Applications Handbook . It includes all of the LI-COR BIO products (Odyssey® family of imaging systems and Pearl® Impulse Small Animal Imaging System, reagents and accessories) plus technical notes, application workflows, and troubleshooting for key applications.

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.

In-Cell Western™ Assay Quality Assessment using Z’-Factor

If you have been following my posts for the past two months or so, you know we have been looking a great deal at In-Cell Western Assays (also called In-Cell ELISAs or plate-based immunofluorescent assays). On April 3, I explained what an ICW Assay is. From there we looked at examples of how In-Cell Westerns are used (studying apoptosis, for IC50 Determinations), seeding plates for In-Cell Westerns), ICW kits LI-COR® offers, and using cells in suspension, plate selection for ICW Assays, and cell lines that have been tested for use with this powerful immunocytochemical assay.

So you are hopefully ready to give this technique a try. When you do, it is important to assess the overall quality and reliability of the assay during In-Cell Western (ICW) assay optimization. The Z’-factor statistic provides a way to evaluate whether or not assay conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’-factor, introduced by Zhang et al., is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data.

Z’-factor experiments are performed on one or more ICW assay plates containing replicate wells designated for background subtraction, negative control samples, and positive control samples. Typically, negative control wells are those in which the cells receive an the appropriate treatment so as to elicit the lowest desired percent response (usually untreated cells); positive control wells are those in which the cells receive an appropriate treatment so as to elicit the maximum desired percent response; background wells are treated the same as the negative control wells, except primary antibody incubation is excluded.

Example of Z' Factor Data Plot

Here are some resources so that you can read more about the Z’-Factor, how to set up the experiments to assess it, and its importance in ensuring you have a high quality, reliable assay method:
Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Fluorescent Cell-Based Assays
In-Cell Western Assay Quality Assessment Using Z’-Factor Data Sheet

Read more about Reproducibility and Precision in In-Cell Western Assays.