No Darkroom? No Problem! Get the LI-COR® C-DiGit® Western Blot Scanner !

C-DiGit Blot Scanner - a Compact Personal Chemiluminescent Imaging System
Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?

FINALLY, image at your convenience. Keep your C-DiGit Blot Scanner on your lab bench, at your desk, or anywhere you choose (look left and see just how small and portable the C-DiGit Western Blot Scanner really is!). It can truly be YOUR personal chemiluminescent Western imager!

The C-DiGit Chemiluminescent Western Blot Scanner maintains the simplicity of film exposures without the mess of the darkroom. You perform all of the same steps, without buying film and spending time in the darkroom. The C-DiGit Scanner gives you a complete digital replacement for film – keeping the advantages of film and eliminating many of the drawbacks – saving you time AND money!

Watch this short video and then visit our website to get your very own C-DiGit Blot Scanner!

NEW! COX IV Primary Antibody Offers Normalization for Low-Expressing Proteins

COX IV Rabbit Monoclonal Primary Antibody, PN 926-42214The COX IV primary antibody can be used for detection of COX IV by Western blot, or as a normalization antibody when performing two-color detection. Its target molecular weight is 17 kDa. Detection of this primary antibody can be achieved with IRDye® Goat Anti-Rabbit or IRDye Donkey Anti-Rabbit secondary antibodies. LI-COR® also carries beta-actin, alpha-tubulin, and beta-tubulin primary antibodies for normalization when performing quantitative Western blots or In-Cell Western Assays.

Western Blot Linearity of COX IV vs. Actin in Cell Lysate

Figure 1. Linearity comparison of COX IV rabbit monoclonal primary antibody (P/N 926-42214) to β-Actin rabbit monoclonal (P/N 926-42210). Primary antibodies were compared by Western blot and detected with IRDye 800CW Goat anti-Rabbit (P/N 926-32211). The COX IV antibody can be used as a mitochondrial loading control and a loading control for normalizing low expressing target proteins. This COX IV primary antibody remains linear with increasing concentrations of lysate, making it ideal for normalization.

COX IV Rabbit Monoclonal Antibody Pack Insert

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 In-Cell Western 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.

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.

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

Seeding Cells in Microplates for In-Cell Western™ Assays – Hints & Tips

In-Cell Western Assays - Fluorescent Immunoassays
One of the first steps in an In-Cell Western Assay experiment is to seed cells into the wells of a tissue culture microplate. Cell density is more important for some cell lines than others. In particular, cells that depend more on extracellular activity for proliferation (such as epithelial cells) are affected to a greater extent by initial growth conditions. There are three factors to consider when seeding cells:

  1. Plates: For most adherent cells that stick to wells tightly (e.g. A431, HeLa, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, such as Nunc P/N 167008. For adherent cells that could detach from wells during In-Cell Western assay wash steps (e.g. NIH/3T3), we recommend Poly-D-lysine coated 96-well microplates.
  2. Cell seeding density: Typically, 15,000 to 40,000 cells are seeded per well. Two to three days are usually required for cells to reach the appropriate confluency, depending on growth rate. Seeding with low cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.
  3. Confluence: To obtain maximal fluorescent signals, complete or near complete confluency is recommended for cells that stick to wells tightly. For cells that adhere loosely to wells, such as NIH3T3, 70% confluency should be used. Please note that cell type and experimental conditions may affect the acceptable level of growth confluency.

The example below illustrates the importance of cell seeding density for A431 cells. As shown in the corresponding graph, cell growth is greatly inhibited when there are too few neighboring cells.

cell seed plate

Graph showing why Seeding Plates for ICW Assays is Important