In-Cell Western Assay
Introduction to In-Cell Western Assays
In-Cell Western (ICW) is a powerful quantitative and qualitative technique that enables the analysis of protein expression in-situ without the hassle of cell lysate sample preparation.
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The In-Cell Western User:
In-Cell Western is 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.
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Uses for In-Cell Western:
- Quantification of protein expression changes in response to drug treatments or genetic manipulations
- Evaluation of protein- protein interactions
- Screening large libraries of compounds in drug discovery and development
- Analysis of protein localization within specific cellular compartments
Example of a typical In-Cell Western workflow
Templates and Protocol
Download these pre-made plate templates and optimized protocol to simplify your In-Cell Western Image Lab Analysis:
Protocol Length Comparison: In-Cell Western vs Traditional Western Blot
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.
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Total Time for In-Cell Western (up to 384 samples)
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Total Time for Traditional Western Blot (up to 24 samples)
Advantages of In-Cell Western Assay vs. Traditional Western Blotting
Advantage 1:
Shorter protocol — no need for time-consuming sample preparation, electrophoresis, and transfer steps.
Advantage 2:
Quantification of multiple targets (up to 3-plex) in numerous samples (up to 384-wells).
Advantage 3:
Protein detection in situ — qualitative and quantitative immunofluorescence.
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 as 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.
Applications & Technologies
Protocol & Templates
Download these pre-made plate templates and optimized protocol to simplify your In-Cell Western Image Lab Analysis:
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Image
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.
Western Blot University
Courses designed to make you a western blotting expert.
