How It Works: A New Approach to Western Blotting

By Bio-Rad Laboratories

Problem: A mainstay in life science research, western blotting is commonly used to assess relative protein expression differences, such as when investigating cellular signaling pathways. Accurate quantification requires careful attention to procedural details and normalization. Traditionally, western blotting is a two-day process with many steps: electrophoresis, transfer, blocking, primary and secondary antibody application and detection. Optimization is often challenging because when blots fail, it’s difficult to determine whether it was a result of the steps leading to antibody binding and detection, or whether the problem was due to the antibody itself.

Solution: One system that may help is the V3 Western Workflow™ from Bio-Rad Laboratories, Inc., which uses stain-free technology to confirm that protein separation and transfer performed as expected and that subsequent immunodetection will result in high quality western blot data. This new system cuts the workflow time in half, while stain-free technology ensures faster, more accurate quantitation of protein expression levels.

The V3 Western Workflow combines Bio-Rad’s TGX Stain- Free™ precast gels, TransBlot® Turbo™ transfer system and the ChemiDoc™ MP imaging system into an integrated solution.

A system like this can deliver considerable time-savings through the electrophoresis and transfer steps. For example, running times on TGX Stain-Free gels are 15 minutes versus 45 minutes for traditional mini gels. Meanwhile, the Trans- Blot Turbo achieves protein transfer in seven minutes, compared to one hour or overnight for traditional tank methods. Transfer efficiency is comparable.

Embedded stain-free technology in the gels adds confidence to the western blotting process by allowing easy quality control. Using the ChemiDoc MP imaging system and TGX Stain- Free gels, researchers can visualize post-electrophoresis gel results in one minute to ensure the sample is not degraded. This represents a significant advance over traditional Coomassie gel staining methods that can take up to two hours to complete and are not western blot compatible. The ChemiDoc MP also allows researchers to check the membrane prior to blot detection and instantly visualize protein transfer. The gel can be subsequently re-imaged to establish if any proteins remain.

Importantly, stain-free imaging enables total protein normalization for more accurate quantitation. Total protein normalization has been shown to be more consistent than conventional housekeeping proteins, which perform variably under certain experimental conditions or sample types. Stain-free technology can therefore reduce the four-hour western blot normalization process by eliminating sequential probing or stripping and reprobing steps necessary for housekeeping protein detection

Whereas faster processes can often come at the cost of data quality, systems like the V3 Western Workflow provide more rapid results while increasing confidence that those results are valid.

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The V3 Western Workflow consists of five steps, with each step enabled by Bio-Rad’s family of blotting instruments and/or products:

1. Protein separation: Bio-Rad’s Criterion™ and Mini-PROTEAN® TGX Stain-Free™ precast gels

2. Visualize protein: ChemiDoc™ MP imaging system

3. Transfer protein: TransBlot® Turbo™ transfer system

4. Verify transfer: ChemiDoc MP imaging system

5. Validate western blot: ChemiDoc MP imaging system







Categories: How it Works

Published In

Appifying the Laboratory Magazine Issue Cover
Appifying the Laboratory

Published: May 5, 2012

Cover Story

'Appifying' the Laboratory

In recent years, research labs have been a notable beneficiary as a variety of highly functional and specialized apps have gained the acceptance and even gratitude of a broad spectrum of laboratory managers, research scientists and bench technologists.
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