Using the Precision Plus Protein™ WesternC™ Standard as a QC Tool at Key Steps in Your Electrophoresis and Blotting Workflow

As a fundamental tool in protein research, it is critical that all steps of the electrophoresis workflow (from running gels to visualizing blots) are performed successfully to ensure accurate, reliable, and reproducible results. Protein standards are often used to monitor electrophoretic separation and transfer efficiency but do not always enable ladder visualization after blot development. Bio-Rad’s Precision Plus Protein WesternC standard enables efficient monitoring of all key electrophoresis and blotting steps, allowing quality control checkpoints throughout the workflow. Precision Plus Protein WesternC standards enable you to:

• Visualize and monitor electrophoretic separation to ensure normal progression
• Assess transfer of western blots
• Visualize the standard and estimate MW of protein samples after chemiluminescence blot development (with use of Precision Protein™ StrepTactin-HRP or StrepTactin-AP conjugates; Figure 1)
• Visualize standards if using fluorescence detection for blots

Tips for Troubleshooting Chemiluminescence Blots

Problem: Bands are too dark upon chemiluminescence development

• StrepTactin dilution was too low for the sensitivity of the chemiluminescence kit used
  • Use a higher dilution of StrepTactin or use a more sensitive chemiluminescence kit*
• The blot was incubated too long with the chemiluminescence substrate
  • Use the recommended incubation time of the substrate kit
• Exposure times were too long

Problem: Bands are too faint upon chemiluminescence development

• StrepTactin dilution was too high for the sensitivity of the chemiluminescence kit used
  • Lower the dilution of StrepTactin or use a more sensitive chemiluminescence kit*
• The blot was not well incubated with the chemiluminescence substrate
  • Make sure that the surface of the blot is well covered with the substrate solution; also, use the incubation time recommended in the substrate kit
• Precision Plus Protein WesternC standard was either over-transferred or under-transferred due to the transfer time used
  • Optimize the transfer time
• Insufficient exposure time. If using a CCD imager, the substrate kit was not optimized for the CCD imaging
  • Use the chemiluminescence kit that has strong signal intensity and long signal duration*

* Such as Immun-Star™ HRP or AP kits or the Immun-Star^™ WesternC™ chemiluminescence kit (optimized for CCD imager).

Fig. 1. Mechanisms by which Precision Plus Protein WesternC or unstained standards work with StrepTactin conjugates.

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. 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.