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Intro to In-Cell Western Assays | Advantages of In-Cell Western Assays | In-Cell Western Workflow and ChemiDoc MP Systems | Image Lab Analysis of In-Cell Western Assays | Templates & Protocol |
Introduction to In-Cell Western Assays
The In-Cell Western Assay (ICW) is both a qualitative and quantitative immunofluorescence assay commonly performed on cultured cells in microplates (optimized for 96-well plates). ICW offers not only enhanced detection and specificity comparable with traditional western blotting, but it also offers higher reproducibility and output comparable to that of immunofluorescence and ELISA. As an immunofluorescence application, ICW allows researchers to use primary antibodies and spectrally distinct conjugated secondary antibodies to detect their proteins of interest in fixed and permeabilized cultured cells. ICW is a rapid and accurate measure of relative protein levels in numerous samples without the need for lysing cells, which prevents possible artifacts. With the elimination of sample prep, SDS-PAGE, and membrane transfer steps used in traditional western blotting protocols, ICW requires less hands-on time. Commonly, secondary antibodies conjugated with near infrared (NIR) fluorescent dyes are used for ICW multiplex detection and normalization but Bio-Rad’s ChemiDoc Imaging System allows utilization of a broader range of fluorescently-labeled secondaries that can be used in tandem with Bio-Rad's ready-to-use hFAB™ Rhodamine Housekeeping Protein Fluorescent Primary Antibodies.
Advantages of In-Cell Western Assay vs. Traditional Western Blotting
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Advantage 1:
Shorter protocol — no need for time-consuming sample preparation, electrophoresis, and transfer steps.
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Advantage 2:
Quantification of multiple targets (up to 3-plex) in numerous samples (up to 384-wells).
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Advantage 3:
Protein detection in situ — qualitative and quantitative immunofluorescence.
In-Cell Western Protocol vs Traditional Western Blotting (Total Protocol Length)
The In-Cell Western Assay is an efficient, time-saving method ideal for those who have already validated the specificity of their antibodies with traditional western blotting or for those interested in overall protein expression without needing to account for molecular weight.
Total Time for In-Cell Western (up to 384 samples)
1. Fix & Permeabilize.
2. Blocking.
3. Immunodetection & Washes.
Total Time for Western Blot (up to 24 samples)
4. Sample Preparation.
5. Electrophoresis.
6. Transfer, Blocking, Immunodetection and Washes.
In-Cell Western Assays Made Easy Using the ChemiDoc MP System
The top reasons why our ChemiDoc MP System is superior to competitor imagers for In-Cell Western Imaging:
- Enables multiplexing of up to 3 targets — unlike methods from competitors that only allow up to 2 targets
- Not constrained to NIR dyes — ChemiDoc MP System has five fluorescent channels
- Provides superior image quality and pairs seamlessly with Image Lab Software for simplistic analysis
- Equipped with ready-to-use controls when combined with our hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies
Find the Right hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibody for Your Experiment
Bio-Rad offers commonly used house-keeping primary antibodies conjugated to a rhodamine dye, making it easy to add to any assay and a natural control.
Image Lab Analysis of In-Cell Western Assays
Image Lab Software simplifies In-Cell Western analysis by taking advantage of its harmonious pairing with any ChemiDoc Imager, making it a powerful yet easy-to-use package for acquisition and analysis of your In-Cell Western Plate. By using the downloadable plate templates provided on this page or creating your own using the Volume Tool, Image Lab Software automatically calculates volume intensity of each well. When coupled with Bio-Rad’s hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies, you can set control wells along with blank wells that are used for background subtraction to calculate adjusted volume intensity. In-Cell Western analysis allows for quantification of multiple targets using spectrally distinct fluorescent dyes and is a quick method for finding relative protein levels in numerous samples.
Templates and Protocol
Download these pre-made plate templates and optimized protocol to simplify your In-Cell Western Image Lab Analysis:
Applications & Technologies
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Western Blotting
Learn about the powerful technique that allows you to positively detect your proteins, estimate quantities, and determine their molecular weights.
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Western Blot Learning Center
The Western Blot Learning Center is a complete reference on all of the steps of western blotting, includes practical theory, protocols, and recommendations on how to make your blots better from experts.
Related Products
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ChemiDoc Imaging Systems
See our stain-free imaging systems that enable immediate visualization of proteins without gel staining and instant verification of protein transfer to blots.
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Image Lab Analysis Software
View the Image Lab Software, an intuitive yet powerful analysis software that is compatible with ChemiDoc Imaging Systems and the GelDoc Go Imaging System.
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hFAB™ Rhodamine HKP Antibodies
Select our hFAB Rhodamine Housekeeping Protein Primary Antibodies that allow one-step detection of common housekeeping proteins such as actin, tubulin, and GAPDH.