Sensitive detection of fluorescence in western blotting by merging images

<div><p>The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.</p></div

Increase in signal intensity by selecting appropriate fluorescent dyes.

<p>Comparison of detection sensitivity among fluorescent dyes. Joined images of dilution series of GST and background area are shown. GST was detected by anti-GST antibody, fluorescent dye conjugated to secondary antibody, and narrow-band filters (Em: 500–520 nm, 570–590 nm, and 665–715 nm). Relative integrated density of 1.56 ng GST signal is shown (mean ± standard error of three independent experiments). Exposure time was 1/1.5 seconds, 1/3 seconds and 1 second in green, yellow and far-red wavelength regions respectively.</p

Reduction in noise signals by narrowing emission wavelength region.

<p>Effects of using narrow-band filters on detection sensitivity. Joined images of dilution series of GST and background area are shown. GST was visualized using anti-GST antibody and fluorescent dyes conjugated to secondary antibody (CF488A, Alexa 546, and Alexa 647). Emission wavelength (Em) of filters and relative integrated density of 1.56 ng GST using general filters (upper lane) and narrow-band filters (lower lane) are shown (mean ± standard error of three independent experiments). Exposure time was 1/3 seconds in the normal filter and 1/7.5 seconds in the narrow-band filter in detecting GFP and Alexa 546, and was 1–1.5 seconds in the normal filter and 1.5 seconds in the narrow-band filter in detecting Alexa 647.</p

Optimized combinations of filter and fluorescence dye in each color wavelength region.

<p>Optimized combinations of filter and fluorescence dye in each color wavelength region.</p

Joined image generated using fluorescence detection method.

<p>Dilution series of recombinant GST detected by our fluorescence method and by the chemiluminescence method. In our method, multiple images are captured using fluorescence microscopy, and joined to make a single image with a wide field and high resolution. The joined image has a broad area and high sensitivity comparable to that of the chemiluminescence method.</p

Increase in signal intensity by selecting appropriate fluorescent dyes.

<p>Comparison of detection sensitivity among fluorescent dyes. Joined images of dilution series of GST and background area are shown. GST was detected by anti-GST antibody, fluorescent dye conjugated to secondary antibody, and narrow-band filters (Em: 500–520 nm, 570–590 nm, and 665–715 nm). Relative integrated density of 1.56 ng GST signal is shown (mean ± standard error of three independent experiments). Exposure time was 1/1.5 seconds, 1/3 seconds and 1 second in green, yellow and far-red wavelength regions respectively.</p