BIOTECHNOLOGY 63
REPRODUCIBILITY
IMPROVING Western blot O
Sam Egel reports on the advantages of IR fluorescence imaging technology
btaining reproducible data from
chemiluminescent
Western blots imaged with film is a difficult task. Te need for consistent results has led to the development of infrared (IR) fluorescence imaging technology that overcomes variability inherent to chemiluminescent detection.
and leads to inconsistent replicates.
The limitations of film Film’s non-linear response to low- and high-intensity signals impacts data integrity. Tese signals do not induce a linear change in optical density (darkness level) on film, limiting the accuracy of quantification.
from the way film records signals using light-sensitive silver halide grains. Photons strike a silver ion in one of these grains and reduce the ion to elemental silver, forming a ‘latent’ image. During development, the elemental silver catalyses the conversion of the grain to dark metallic silver. Optical density increases as additional silver grains are converted to metallic silver. However, the relationship between the number of photons that strike an image and the number of grains converted to silver is not always linear. Low-intensity signals do
Fig. 1. Chemiluminescent signals are inconsistent between identical blots. An antigen dilution series was detected on six replicate blots. Substrate was applied to all blots in parallel, and all were exposed simultaneously to the same sheet of film for five minutes. Signal strength is still inconsistent between blots
Limitations of
chemiluminescence make it difficult to achieve consistent Western blot results. Chemiluminescence is an enzymatic reaction that generates light to indicate the presence of a target antigen. Te rate of this reaction varies over time, so light production is not constant. Blots imaged at different stages in the reaction, due to delays such as waiting for the darkroom, will be inconsistent. Even identical blots imaged at the same time can produce inconsistent results (see Fig. 1). Accuracy is further
compromised by stripping and re-probing chemiluminescent blots to detect multiple targets. Stripping can cause loss of antigen from the membrane,
Tis non-linear response, called ‘reciprocity failure’, stems
not produce photons rapidly enough to irreversibly activate silver grains. Activated grains are unstable; they must absorb multiple photons to create a latent image, or they will revert to the unactivated state and a latent image will not form, even with long exposures. Tis is called ‘low-intensity reciprocity failure’ and is demonstrated in the ‘toe’ region of the graph in Fig. 2. Strong signals cause film
response to plateau and become
Fig. 2. Non-linear response of film to low-intensity and high- intensity signals. Sigmoidal curve showing Optical Density versus the Log of Relative Exposure for film. The regions labelled ‘toe’ and ‘shoulder’ indicate the limitations of film for documentation of faint and strong signals, respectively
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