Film vs. Digital Western Blot Imaging

Publication Requirements

Need another reason to get out of the darkroom?

Several journals have recently revised their guidelines for publication, providing guidance around reproducibility and quantitation, requirements for data reporting, and specific recommendations on digital imaging of gels and quantitative western blots.

• 

  Nature

• 

  Science

• 

  Journal of Biological Chemistry

Publication requirements are changing. Are your blots ready?

• 1

  "House-keeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression"*

  Solution: Use Total Protein Normalization (TPN) instead of house-keeping proteins

  • Bio-Rad's stain-free workflow makes TPN easy! This technology is proven with over 100 publications, allows for improved accuracy and will make your data more reliable

• 2

  "Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range"*

  Solution: Use a digital imaging system with up to 4 logs of dynamic range

  • Bio-Rad's ChemiDoc System — an imager with the sensitivity of film
  • Simple image generation and quantitation using Image Lab Software

• 3

  "A description of the data supporting the specificity of all antibodies is required"*

  Solution: Fully validated antibodies

  • Bio-Rad's PrecisionAb Antibodies offer documented specificity and validation protocols
  • Phospho-Specific PrecisionAb Antibodies are validated in treated and untreated cell lysates and by dephosphorylating western blot membranes

* Revised guidelines for authors from The Journal of Biological Chemistry

Total Protein Normalization provides accurate quantitation of target proteins.

Normalizing western blots with housekeeping proteins (HKPs) requires time, money, and additional sample to validate HKP expression and linearity. Total protein normalization using stain-free technology and a ChemiDoc Digital Imaging System allows normalization across a wide dynamic range. Get better, more publishable data without lengthy optimization.

Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10–50 µg of HeLa cell lysate.

Design your Western Blotting Experiments with Confidence

As an editor of several journals, Professor Aldrin V. Gomes routinely observes western blots that are not properly conducted. The generally low quality of western blotting data has led scientists to question whether we can trust protein quantification when using western blots. Reducing errors and misinterpretations requires standardized procedure and proper controls. Dr. Gomes discusses the following key factors for high-confidence western blot data:

1. Quality of the primary and secondary antibodies and the blocking reagent
2. Proper target protein controls
3. Choice of total protein loading control vs. housekeeping protein loading control to normalize the target protein measurement

Prof. Gomes also presents tips to reduce the overall time required for first-rate western blots, and how to minimize the pain of going through a long procedure.

• Reliable Quantification of Western Blot Data

  Based on the article "A Defined Methodology for Reliable Quantification of Western Blot Data". Dr. S.C Taylor provides a direct comparison between results generated from camera-based imaging technologies and film using identical blots. Learn common pitfalls associated with film use and densitometric analysis.

  This video is based on the article published by S.C. Taylor et al. entitled "A Defined Methodology for Reliable Quantification of Western Blot Data" (DOI 10.1007/s12033-013-9672-6).

• Designing a Quantitative Western Blot Experiment to Avoid Common Pitfalls

  The quantitative western blot methodology is described with a focus on solid experimental design and data analysis to achieve reproducible, publication quality results in the shortest time frame.

  This video is based on the article published by Drs. Taylor and Posch entitled: "The Design of a Quantitative Western Blot Experiment".

• Quantitative Western Blotting: Optimization is the Key

  Dr. S.C. Taylor details the common errors associated with performing quantitative western blots and how they can be addressed through a rigorous optimization methodology. Each step of the quantitative western blot workflow is discussed with a focus on optimization to assure quantitative, reproducible final data that reflect the experimental conditions.

Testimonial

Leukemia researcher Ernesto Diaz-Flores (University of California, San Francisco) describes how the ChemiDoc Digital Imaging System helped him improve his western blot imaging, enabling him to identify relevant therapeutic targets. An avid photographer, Prof. Diaz-Flores also talks about the links between his art and his science and the technologies that help him achieve his goals in both fields.

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