imaging of chemiluminescentwestern blots: comparison...
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Imaging of Chemiluminescent Western Blots:Comparison of Digital Imaging and X-ray Film
Genome Research Centre, Hong Kong UniversityJune 5, 2009Hong Kong
• Western Blotting
• Film detection
• CCD Imaging
• Optimized solution: CCD detection, brighter substrates, chemiluminescent markers
• Multiplex Fluorescent Blotting
Western Blotting
• Towbin et al (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-5354.
– Original band pattern retained and no loss in resolution
– membrane-bound antibody-protein complex is detected by a secondary antibody
• Radioactively labeled
• Conjugated to FITC
• Conjugated to horseradish peroxidase (HRP), in conjuction with a substrate that produces a luminescent signal.
– Limit of detection is 100 picograms
Chemiluminescent Detection and Film
• Chemiluminescent detection is widely popular (Towbin et al. 1979; Kricka 1991, Rongen et al. 1994, Beck et al. 1990).
– High sensitivity, large dynamic range, rapid results, low cost, and safety
– X-ray Film: traditional detection
• Researcher needs
– Quantitative Data
• Better dynamic range
• Longer lasting signals
– Sensitive detection
– Reproducible
– Easy to use
• Bracketing
• Molecular weight determination
• Limited substrate signal duration
Detection comparison: Film vs. CCD
• Detection reagents: ECL vs. Western C+
• CCD imaging systems performance: ChemiDoc vs VersaDoc vs Film
• Quantitative Data: Linear dynamic range
Longer Lasting Signals: Better Detection Reagent
• Brighter starting signal
• Longer lasting signal
– Wider and more linear dynamic range
– Improved sensitivity
CCD Imaging
• Gel Doc for DNA application, 1995
– No more dark rooms
– Quantitative vs visual inspection
– No more films
• ChemiDoc for Western Blotting
– Similar concerns resolved
– Easy to use
– Reproducible
Limit of DetectionFilm and CCD detection of Western Blots
• Film
– Long exposures needed for low signal intensities
– High signal intensities saturate quickly
– Overexposure causes bands in close proximity to merge
• CCD
– Captures broader range of signal intensities in a single exposure without saturation (quantitative dynamic range)
– Significantly lower limit of detection (LOD) for improved sensitivity
Linearity of Film and CCD Chemiluminescent Western Blot Detection
• Film detection of Western blot
– Sigmoidal with narrow linear response
• CCD detection of Western blot
– Broader linear response
No Loss in Performance
• Film detection of Western blot
– Sigmoidal with narrow linear response
• CCD detection of Western blot
– Broader linear response
Designed for ease of use and ability to
support a wide array of applications, it is a
perfect fit for individual laboratories or
multiuser core facilities.
• High sensitivity and quantitative images
• Reproducible
• Flexible and easy to use platform
• Quality optics
• Trusted name
ChemiDoc XRS+ System
Film
ChemiDoc XRS
Time (seconds)15
Molecular ImagerSystem
Quantitative Dynamic RangeChemiDoc XRS+ System vs. Film
Figure 1. Harta LED test targets imaged with Bio-Rad's Molecular Imaging ChemiDoc XRS Systems, as well as exposed to
film, for 15 seconds. The Harta target produces illumination that range from bright to very faint. It is controlled tool for testing
the dynamic range of any luminescence detection system, without the variables usally introduced during sample prep,
electrophoresis, and blotting. The ChemiDoc XRS System displays a high linear dynamic range, enabling it to see all 7out of 9
spots in 15 seconds, without saturating the brightest signal. This quantitative data is due to the 16,000 electron well capacity of
the CCD and low 4.5 electron noise. The high quantum efficiency of the CCD also enables the system to detect signal fast,
avoiding building up of background noise. In 15 seconds, film also detected 7 out of 9 spots, and the 4 brightest spots are
saturated. This illustrates the limited dynamic range of film. While film is a good tool to see the absence or presence of a
sample, it is not quantitative.
Figure 2. Regression curve of the 15 second exposure on the ChemiDoc XRS System and film. The R-Squared
value of a regression curve is an indicator of its quality; a desirable value is as close to 1.00 as possible. The R-
Squared value for the ChemiDoc 15 second exposure for 7 spots on the Harta target is 0.999777. The R-
Squared value for the 15 second exposure on film for 7 spots on the Harta target is 0.658073.
Linear Dynamic RangeChemiDoc XRS System vs. Film
ChemiDoc XRS System Correlation Coefficient = 0.999777
Film Correlation Coefficient = 0.658073
Immun-Star WesternC Kit
• 24-hour light emission
• Femtogram-level sensitivity
• HRP based system is compatible with all types of HRP conjugated antibodies
• 12 month shelf life at room temperature
Chemiluminescent Signal/Background Over
Time
0
2
4
6
8
0 50 100
Time (minutes)
Mean Signal/BKG
ECL+
Upstate
WesternC
Other Bio-Rad Chemiluminescent Kits
Colorimetric Detection: Amplified Opti-4CN Kits
Secondary Antibodies: GAR, GAM, GAH, Protein A, Protein G
Precision Plus Protein WesternC Standard
• Matching band pattern on the gel, membrane and blot
• A total of ten protein bands spanning 10 to 250 kD (10, 15, 20 25, 37, 50, 75, 100, 150 and 250 kD)
• 3 reference bands (25, 50 and 75 kD) are prestained pink while all other bands are prestained blue
• R2 of the Rf vs log (molecular weight) plot > 0.99 guarantees high accuracy in estimating molecular weigh
R2 = 0.9968
0
0.5
1
1.5
2
2.5
3
0 0.2 0.4 0.6 0.8 1
Rf
log MW
ChemiDoc XRS+ and Western C
• Limit of detection is improved by 2-fold
Signal is brighter and lasts longerBulletin 5809: Imaging of Chemiluminescent Western Blots: Comparison of Digital Imaging and X-ray Film
Color Standards and Chemi Samples
Image
• Easily determine the molecular weight of chemi samples
Merge Analyze
Multiplex Fluorescent Blot using Quantum Dots
• Improve linearity when comparing low and abundant samplesBulletin 5809: The Dynamic Range Effect on Protein Quantitation in Polyacrylamide Gels and on Western Blots
Workflow Applications
Ethidium Bromide
SYBR Safe
SYPRO Ruby
Flamingo
Coomassie
Silver Stain GFP and Coomassie
Multi-Fluorescent Western Blot
• Bulletin 5685: Effect of PMA on Phosphorylationof Cx43: A Quantitative Evaluation Using Blotting with Multiplex Detection
Phosphorylated Cx43
Total Cx43
Tubulin
Merged