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

No loss in sensitivity

ChemiDoc XRS+ Film

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

Live Image Acquisition

• Remove the guesswork with time exposure

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