Using Precision Plus Protein™ WesternC™ Standards to Validate Major Steps in the Western Blotting Workflow

Introduction

Western blotting, although a commonly used technique, involves a time-consuming multi step process. The whole western blotting workflow can take up to three days to complete. Thus, having a tool to monitor each of the major steps in the western blotting workflow reduces the chances of finding out at the very end of the workflow that an experiment was not successful. Bio-Rad’s Precision Plus Protein WesternC standard enables you to check the progression and success of all the major steps in the western blotting process. This protein standard is unique in that it offers the ability to visualize and monitor electrophoretic separation, assess transfer of proteins to your membrane, and visualize and estimate the molecular weight of your target after blot development with a StrepTactin-HRP or AP conjugate. This protein standard can be used regardless of your detection method* (colorimetric, chemiluminescent or fluorescent). The following workflow illustrates the use of Precision Plus Protein WesternC standard with a suite of Bio-Rad products, all of which minimize the time to results for your western blotting workflow.

*StrepTactin-HRP or StrepTactin-AP conjugate is needed for colorimetric and chemiluminescent blot development, but not for fluorescence development. The WesternC standards have dyes that fluoresce under green and red wavelengths.

Monitor Your Western Blotting Workflow with Precision Plus Protein WesternC standards

WesternC protein standards have dual-colored prestained bands that enable you to monitor electrophoretic separation. The WesternC standards are shown below after electrophoretic separation on a 4–20% Mini-PROTEAN^® TGX™ gel, which allows protein separation in as little as 15 minutes. Once the ten standard bands are separated and visualized, you can move onto the next step of the workflow.

WesternC protein standards also enable easy monitoring of transfer process, providing a broad molecular weight range, from 10 to 250 kD. Additionally, the dual-color feature of the standard provides a quick way to look at the orientation of your blot. The WesternC standards are shown below on a PVDF membrane after a transfer using the Trans-Blot^® Turbo™ transfer system, which enables protein transfers in as little as three minutes. Visualization of all ten bands of the standard on your membrane indicates that proteins have transferred, and the blot can be moved to the next step in the western blotting workflow.

After developing the blot via chemiluminescence using Precision Protein^® StrepTactin-HRP or -AP conjugates or fluorescence (no conjugate needed), one can then confirm the target protein(s) by molecular weight. The WesternC protein standards are shown below (left) on a blot developed via chemiluminescence (using the Precision Protein StrepTactin-HRP conjugate) and are shown (right) on a blot developed via fluorescence. The blue-stained WesternC standard bands fluoresce in the red range (10, 15, 20, 37, 100, 150, 250 kD) and the pink bands (25, 50, 75 kD) fluoresce in the green range; the fluorescent blot images were taken using Bio-Rad’s VersaDoc™ MP 5000 imaging system and overlaid. The target proteins (soybean trypsin inhibitor, 22 kD; and human transferrin, 80kD) in all sample lanes are loaded in a dilution series from 1ng to 400 ng.

Ensure Success Before Moving on to Your Next Step in the Electrophoresis/Blotting Process

Precision Plus Protein WesternC standards is a combination of prestained and unstained standard that enables you to:

• Visualize and monitor the electrophoretic separation to ensure it is progressing normally
• Assess transfer in western blots
• Visualize the standard and estimate MW of protein samples after blot chemiluminescence development (with use of Precision Protein StrepTactin-HRP or -AP conjugate
• Visualize standard if using fluorescence blot detection (no conjugates needed)

Conclusion

Having a tool to ensure success before moving onto the next step in the electrophoresis and blotting workflow is important because one can avoid having to repeat the lengthy process due to the failure of one step. Precision Plus Protein WesternC standard can be used to quality control all the major steps of your blotting workflow, ensuring the success of each step before you move on to the next. Along with the WesternC standards, Bio-Rad also offers a whole portfolio of products, including the TGX™ line of fast running gels and the Trans-Blot Turbo transfer system, that streamline the time spent on western blotting experiments.

Precision Plus Protein standards are sold under license from Life Technologies Corporation, Carlsbad, CA, for use only by the buyer of the product. The buyer is not authorized to sell or resell this product or its components. StrepTactin is covered by German patent application P 19641876.3. Bio-Rad Laboratories, Inc. is licensed by Institut für Bioanalytik GmbH to sell these products for research use only.