Optimization of Western Blots Detecting Proteins Synthesized Using Cell-Free Expression

Cell free protein expression has become a convenient method for generating small amounts of protein for a variety of applications (e.g. protein:protein interactions, protein: nucleic acid interactions). One of the most popular formats is based on rabbit reticulocytes. Using a mammalian based system offers researchers the optimal environment for the expression of functional proteins (e.g. correctly folded or modified).

When using a rabbit reticulocyte based translation system to express your protein of interest followed by detection using Western blotting there are a few critical parameters to keep in mind in order to optimize your experimental data. Excessive background can be caused by several factors.

  • One of the most common mistakes is loading too much lysate on the gel. Typically loading between 0.5µl and 2µl of lysate should provide an adequate signal to detect.
  • Be sure not to use a secondary antibody directed against rabbit proteins, since it will react with the lysate components.
  • If you are experiencing background when using horseradish peroxidase detection, the peroxidase activity from the hemoglobin maybe the issue. To prevent the hemoglobin from reacting with the substrate during detection, soak the membrane in TBS containing 10% ImmunoPure Peroxidase Suppressor (Pierce Cat # 35000) for 30 minutes prior to blocking.

For additional information about cell-free protein expression check out the Protein Expression Chapter of the Promega Protocols and Applications Guide.

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Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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