Comparing X-Ray Film and Digital Imaging Systems
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|Advantages of Digital Imaging Systems||ChemiDoc Imaging System||Publication Requirements||Designing Experiments||Resources||Related Products|
X-ray film has long been considered the gold standard of sensitivity in western blotting, but the broad dynamic range enabled by digital imaging systems allows for accurate quantitation of a range of signal intensities. With recent technological advances, can researchers finally have the best of both worlds?
Advantages of Digital Imaging Systems
Key benefits to using this technology include:
- Wide dynamic range = > 4 logs
- Digital documentation
- Environmentally friendly — no chemicals!
- No costly consumables (film)
- Initial capital investment typically recovered
in as little as 1 year
See the Data
The sensitivity of film, with a broad dynamic range and superior signal-to-noise.
ChemiDoc MP Imaging System: Imagine No Compromises
With the ChemiDoc MP Imaging System, the superior quantitation of digital imaging meets the sensitivity of film. This best-in-class system images and analyzes gels and western blots. It is designed for multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation, and stain-free technology imaging needs.
ChemiDoc Imaging System
Better Options. Better Results.
Total Protein Normalization
Learn how to properly design a quantitative western blotting experiment and faithfully compare protein expression levels, without concern for changing or overexpressed HKP’s.
See Data »
Save money on consumables and reduce environmental waste without compromising on performance. Get the sensitivity of film and the convenience of digital documentation.
See Data »
Save sample, avoid the errors commonly associated with stripping, reprobing, and cutting blots and answer more complex biological questions by optimizing your experiments to fluorescence.
See Data »
Need another reason to get out of the darkroom?
Several journals have recently revised their guidelines for publication, providing guidance around reproducibility and quantitation.
Publication requirements are changing. Are your blots ready?
"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
"Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range"*
Solution: Use an 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
"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
Total Protein Normalization provides accurate quantitation of target proteins.
Normalizing 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 the ChemiDoc or ChemiDoc MP System allows normalization across a wide dynamic range. Get better, more publishable data without lengthy optimization.
Learn more about Stain-free Technology
This article focuses on the theory and concepts surrounding stain-free technology as well as other traditional methods of protein staining.
Visualize proteins on gels and blots without staining and destaining steps, and use total protein normalization for quantitative western blots.
Bio-Rad's V3 Western Workflow Incorporates TPN for Normalization of Western Blot Data
V3 Western Workflow includes:
- Stain-free visualization of total proteins on gel after electrophoresis
- Quantification of total proteins in a lane after transfer
- Normalization of bands to total proteins by specialized software
Find out why these 3 steps are crucial to generating quantitative western blot data.
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:
- Quality of the primary and secondary antibodies and the blocking reagent
- Proper target protein controls
- 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.
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.
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.
Leukemia researcher Ernesto Diaz-Flores (University of California, San Francisco) describes how the ChemiDoc 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 technologies that help him in both fields.
Get answers to your questions about imaging
Bio-Rad provides instructional videos for Image Lab Touch Software 2.4, covering the primary applications of the ChemiDoc MP Imaging System. Topics include, logging in, Smart Tray Technology, capturing your image, and exporting data.
Bio-Rad provides tutorials for basic acquisition and advanced analysis use of Image Lab Software 6.0.1. Topics include, densitometric analysis, molecular weight, normalization, purity assessment, and more!
Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.
(PDF 7.9 MB)
Details on blotting technology, available products, and methods, plus tips, techniques, and troubleshooting.
(PDF 6.4 MB)
A guide to polyacrylamide gel electrophoresis and protein detection, including theory, product selection, protocols, and more.
ChemiDoc MP System Brochure
(PDF 1.26 MB)
Explore the features and benefits of the new high-end imaging system for the best fluorescence and chemiluminescence detection.
Stain-Free Approach to Western Blotting
(PDF 864 KB)
Learn how to use stain-free technology for total protein normalization as an alternative to the standard blot normalization process.
V3 Western Workflow Brochure
(PDF 593 KB)
See how the V3 Western Workflow compares to traditional western blotting workflows.
Learn about the methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.
Read a summary about a complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.
Mini-PROTEAN TGX Stain-Free Precast Gels for polyacrylamide gel electrophoresis (PAGE) allow for fast run times, high transfer efficiency, and rapid detection of proteins with stain-free enabled imagers.
These premium antibodies are lab-validated using strict testing criteria to ensure superior performance in western blotting detection.
The Trans-Blot Turbo System is a rapid protein transfer apparatus that can transfer protein to membrane in as little as 3 minutes. Trans-Blot Turbo Transfer Packs provide greater transfer efficiency in less time.
Prestained and unstained molecular weight standards for protein electrophoresis applications including SDS-PAGE, western blotting, 2-D PAGE, and isoelectric focusing (IEF).
Clarity family of Western ECL Substrates provides simple, high-performance solutions for all your western chemiluminescence needs.
SureBeads Protein G Magnetic Beads enable fast, easy, consistent immunoprecipitation without centrifugation.
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