Western blotting is an analytical method that is commonly used for the identification and quantification of specific proteins in a biological sample (Gallagher and Wiley, 2018).
Traditionally, a target protein is interrogated by antigen-specific primary antibodies that are then probed by secondary antibodies conjugated to either Alkaline Phosphatase (ALP) or Horseradish Peroxidase (HRP) and followed by chemiluminescent or colorimetric detection.
There continues to be growing interest in fluorescent protein (Western) blotting employing primary or secondary antibodies labeled with a fluorophore to perform non-enzymatic detection of protein expression because of the improved signal-to-noise and multiplexing capabilities.
Further benefits of fluorescent blotting include accurate quantitative analysis with broader dynamic range and high linearity in addition to excellent signal stability over time, reducing or eliminating the requirement to strip and reprobe.
Methods and materials
- Membrane: PVDF Millipore Immobilon-FL PVDF
- Imaging system: UVP ChemStudio
- Wash buffer: TTBS (Tween (0.1 % Tris Buffered Saline)
- Biotium’s TrueBlack WB blocking kit, Cat#23013 (blocking buffer and antibody diluents)
- Primary antibodies (0.1 μg/ml Primary Mouse or rabbit anti-tubulin)
- HeLa lysate. The total protein concentration is determined by BCA assay. The lysis buffer contains 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 % TritonX-100, pH 7.5
- Secondary antibodies goat-∝-rabbit IgG – CF 680R or goat-∝-mouse IgG – CF 770®
By utilizing a serial dilution of 10 μg, 7.5 μg, 5 μg, 2.5 μg, or 1 μg HeLa cell lysate protein was added into micro sample tubes to total 10 μl. According to manufacturer’s instructions (Biotium MnS Total Protein Prestain kit), lysate dilutions were pre-stained lysate for 30 minutes with CF680T or CF770T for 30 minutes.
Using a dry temp lock, loading buffer (4X LDS loading buffer)+ DTT was added to samples and heated for 3 minutes at 95 °C. Samples were loaded on a 4-12 % Bis-tris SDS-PAGE gel and separated at the constant voltage at 200V for 30 minutes.
Using an automated western blotting instrument according to manufacturer instructions, the separated proteins were then electrotransferred to a PVDF membrane at 20V for 1 hour.
Note. There are a variety of alternatives including semi-automated and manual strategies (Gallagher and Wiley, 2018).
Blots were processed according to the following sequence:
- Blocking, 4 °C 1 h
- Primary Ab binding, 4 °C 16 h
- Washing, 4 °C 5 x 10 min
- Secondary Ab binding, 4 °C 2 h
- Washing, 4 °C 5 x 10 min
The UVP ChemStudio Imaging System and the integrated 660/787 nm lasers were utilized for fluorescent imaging. Images were composited with VisionWorks Touch software and were collected automatically. The processed blot was briefly positioned on the sample plate.
One-touch automated software set the optimal emission and excitation wavelengths, focus, and exposure for optimum signal without saturation and automatically pseudo-colored to show the two different wavelengths and compositing, in addition to saving the original raw data images. The original unaltered image was archived once collected and a copy was utilized for the image analysis.
Results and discussion
The multiplex imaging capabilities of the UVP ChemStudio Imaging System are shown in Figure 1, specifically separating out the signal of both Biotium CF680R and CF 770 fluorescence channels. Fluorescent western blotting applications provide a number of advantages over chromogenic or chemiluminescent visualization.
Most significantly, fluorescent labels enable multiplexing so that multiple proteins in a sample can be detected and analyzed simultaneously, on a single protein blot. In particular, for quantitative imaging, fluorescent labels provide extremely low background and a high signal-to-noise ratio.
The combination of 660 nm and 787 nm NIR excitation laser light sources with the UVP ChemStudio Imaging System with internal blue, green, red narrowband LEDs supplies a complete range of excitation light wavelengths.
It also provides fast, high-resolution image capture through the utilization of deeply cooled 815 8.1 mpx with NIR and QE enhanced 615 3.2 mpx cameras and f/0.95 low light lenses.
Figure 1. Protein blot stained with Biotium MnS Total Protein Prestain kit CF770T and Primary Rabbit anti-Tubulin/ goat-∝-rabbit IgG – CF 680R and visualized with the ChemStudio using the NIR 660/787 lasers set.
The UVP ChemStudio Imaging System supplies a significant advantage compared to laser scanning imaging systems where imaging times range from 3 to 5 minutes, as its imaging times are between 1 to 5 seconds for fluorescent Western blotting applications.
Fluorescent Western blot imaging with the UVP ChemStudio Imaging System is a quick and efficient process that allows researchers to achieve detection of multiple proteins on the same immunoblot to produce high resolution, publication-ready images.
References and Further Reading
Gallagher, S.R. and Wiley, E.A. Current Protocols: Essential Laboratory Techniques. Wiley, 2018
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