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Application Note FP-176


             Multiplex Fluorescent Western Blot Detection
               Using the BioSpectrum Imaging System
                             Abhishek Trikha, BVSc & A.H , M.S. and Samet Serdar Yildirim, MSc
                                                  UVP LLC, Upland, CA


Introduction
Western blotting is a commonly used analytical
technique for the identification and quantification of
specific proteins in a biological sample. Traditionally, a
target protein is interrogated by antigen-specific
antibodies which are then probed by secondary
antibodies conjugated to either HRP or ALP and
followed by colorimetric or chemiluminescent detection.

Fluorescent western blotting employs secondary
antibodies labeled with a fluorophore to perform direct,
non-enzymatic detection of protein expression. On an
immunoblot which is incubated with two different
primary antibodies from different species and then
probed with CyDye™-tagged secondary antibodies for
detection (Figure 1), the two-color multiplexing abilities
of the BioSpectrum® Imaging System with the BioLite™
Xe MultiSpectral Light Source allow for detection of
multiple proteins.

Additional benefits of fluorescent blotting include
excellent signal stability over time as well as accurate
quantitative analysis with broader dynamic range and
high linearity, reducing or eliminating the need to strip
                                                                            Figure 1. Schematic of Fluorescent Western Blotting
and re-probe.

Materials and Methods                                                    Imaging

Sample Preparation and Western Blotting                                  The BioSpectrum Imaging System with the BioLite Xe
                                                                         MultiSpectral Light Source (UVP, LLC) was used for
Two-fold dilutions of normal rabbit and mouse serums                     fluorescent imaging (Figure 3). Images were processed
(Jackson Immuno Research Laboratories) were                              with VisionWorks®LS Image Acquisition and Analysis
separated by SDS PAGE on 4-12% acrylamide gels                           software (UVP, LLC) to remove background intensity
(Invitrogen). The separated proteins were then                           and composite the pseudocolored images.
transferred to nitrocellulose membranes.1
                                                                         Briefly, the processed blot was positioned on the sample
Blots were simultaneously probed with goat-∝-rabbit IgG                  platen. Utilizing software presets, the excitation and
– Cy3® and goat-∝-mouse IgG – Cy5® (GE Healthcare                        emission wavelengths were selected, the lens aperture
Life Sciences) secondary antibodies at 1:1250                            was set at f/1.2, and the image was focused. Exposure
concentration in Western Breeze blocking buffer                          time was adjusted for maximal signal without saturation
(Invitrogen) for one hour incubation at room                             and ranged from 1 to 5 seconds, depending on the
temperature. Blots were washed in secondary antibody                     sample and filter set (Table 1).
incubations with 4 x 5 minutes in tris-buffered saline
(PBS) containing 0.1% Tween 20.


                           UVP, LLC | Upland CA | (800) 452-6788 | (909) 946-3197 | info@uvp.com| www.uvp.com
                       Ultra-Violet Products Ltd. | Cambridge UK | +44(0)1223-420022 | uvp@uvp.co.uk | www.uvp.com
Focal Points Application Note                                                                                      Page | 2

  a)                                                           b)




                              Cy3                                                           Cy5

                               c)




                                                 Multiplexed Cy3 and Cy5

                                                                                               ®
 Figure 2. Multiplex Fluorescent Western Blot Detection with CyDyes™ using BioSpectrum Imaging System
 a) Rabbit IgG probed with Cy3–tagged goat anti-rabbit IgG b) Mouse IgG probed with Cy5-tagged goat anti-mouse IgG c)
 Multiplex fluorescent detection of two fold serial dilution of mouse and rabbit serum proteins probed with Cy3–tagged goat
 anti-rabbit IgG and Cy5-tagged goat anti-mouse IgG, respectively, on same immunoblot. CyDyes were imaged using a one
 second exposure with the BioSpectrum Imaging System.


Once acquired, the original unaltered image was
archived and a copy was used for image analysis. The                Fluorescent western blotting applications offer many
image was adjusted to globally remove background                    advantages over chemiluminescent or chromogenic
intensity and contrast, and was pseudocolored green                 visualization. Most significantly, fluorescent labels
and red using VisionWorksLS Software to indicate Cy3                permit multiplexing so that several proteins in a sample
and Cy5, respectively.                                              can be detected and analyzed at the same time and on
                                                                    a single protein blot. Fluorescent labels in particular
                                                                    offer very low background and a high signal-to-noise
                                                                    ratio for quantitative imaging.
         Dye        Excitation         Emission
         Cy3         525BP45           605BP50                      The combination of the BioSpectrum Imaging System
         Cy5         630BP30           730BP40                      and the BioLite MultiSpectral Light Source provides not
                                                                    only a full range of wavelengths for excitation light but
 Table 1. Filters used for NIR blotting with 680 and 770
                                                                    also rapid high resolution image capture through the use
 to 800nm fluorescent tags.
                                                                    of deeply cooled MegaCam 810 and OptiChemi™ 610
                                                                    cameras and low light lenses.
Results and Discussion
                                                                    Imaging Time
Figure 2 illustrates the multiplex imaging capabilities of
the BioSpectrum Imaging System, specifically                        Compared to laser scanning imaging systems where
separating out the signal of both Cy3 and Cy5 color                 imaging times range from 3 to 5 minutes, the
channels.                                                           BioSpectrum Imaging System provides a significant
                                                                    advantage with imaging times ranging from 1 to 5
                                                                    seconds for fluorescent Western blotting applications.
Focal Points Application Note                                                                          Page | 3



Conclusion                                                 References
Fluorescent Western blot imaging with the BioSpectrum        1.   Gallagher, S.R. and Wiley, E.A. Current
Imaging System and BioLite MultiSpectral Light Source             Protocols: Essential Laboratory Techniques.
is a fast and efficient process for enabling researchers          Wiley, 2012
to achieve simultaneous detection of multiple proteins
on the same immunoblot and generate high resolution,
publication-ready images.




 Figure 3. BioSpectrum Connected to the BioLite
 Together, the BioSpectrum and BioLite are a powerful
 combination that are able to specifically excite and
 illuminate at wavelengths from 365 to 765nm and read
 emissions from 400 to 850nm. Use of up to eight
 excitation and five emission wavelengths are possible
 in a single experiment.

 The BioSpectrum 810 and BioSpectrum 610 are
 recommended for imaging multiplex fluorescent
 Western blots.
Focal Points Application Note                                                                                 Page | 4
BioSpectrum and VisionWorks are registered trademarks of UVP, LLC. BioLite and OptiChemi are trademarks of UVP, LLC.
All other registered trademarks or trademarks are the property of their respective owners.

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Multiplex Western Blot

  • 1. Application Note FP-176 Multiplex Fluorescent Western Blot Detection Using the BioSpectrum Imaging System Abhishek Trikha, BVSc & A.H , M.S. and Samet Serdar Yildirim, MSc UVP LLC, Upland, CA Introduction Western blotting is a commonly used analytical technique for the identification and quantification of specific proteins in a biological sample. Traditionally, a target protein is interrogated by antigen-specific antibodies which are then probed by secondary antibodies conjugated to either HRP or ALP and followed by colorimetric or chemiluminescent detection. Fluorescent western blotting employs secondary antibodies labeled with a fluorophore to perform direct, non-enzymatic detection of protein expression. On an immunoblot which is incubated with two different primary antibodies from different species and then probed with CyDye™-tagged secondary antibodies for detection (Figure 1), the two-color multiplexing abilities of the BioSpectrum® Imaging System with the BioLite™ Xe MultiSpectral Light Source allow for detection of multiple proteins. Additional benefits of fluorescent blotting include excellent signal stability over time as well as accurate quantitative analysis with broader dynamic range and high linearity, reducing or eliminating the need to strip Figure 1. Schematic of Fluorescent Western Blotting and re-probe. Materials and Methods Imaging Sample Preparation and Western Blotting The BioSpectrum Imaging System with the BioLite Xe MultiSpectral Light Source (UVP, LLC) was used for Two-fold dilutions of normal rabbit and mouse serums fluorescent imaging (Figure 3). Images were processed (Jackson Immuno Research Laboratories) were with VisionWorks®LS Image Acquisition and Analysis separated by SDS PAGE on 4-12% acrylamide gels software (UVP, LLC) to remove background intensity (Invitrogen). The separated proteins were then and composite the pseudocolored images. transferred to nitrocellulose membranes.1 Briefly, the processed blot was positioned on the sample Blots were simultaneously probed with goat-∝-rabbit IgG platen. Utilizing software presets, the excitation and – Cy3® and goat-∝-mouse IgG – Cy5® (GE Healthcare emission wavelengths were selected, the lens aperture Life Sciences) secondary antibodies at 1:1250 was set at f/1.2, and the image was focused. Exposure concentration in Western Breeze blocking buffer time was adjusted for maximal signal without saturation (Invitrogen) for one hour incubation at room and ranged from 1 to 5 seconds, depending on the temperature. Blots were washed in secondary antibody sample and filter set (Table 1). incubations with 4 x 5 minutes in tris-buffered saline (PBS) containing 0.1% Tween 20. UVP, LLC | Upland CA | (800) 452-6788 | (909) 946-3197 | info@uvp.com| www.uvp.com Ultra-Violet Products Ltd. | Cambridge UK | +44(0)1223-420022 | uvp@uvp.co.uk | www.uvp.com
  • 2. Focal Points Application Note Page | 2 a) b) Cy3 Cy5 c) Multiplexed Cy3 and Cy5 ® Figure 2. Multiplex Fluorescent Western Blot Detection with CyDyes™ using BioSpectrum Imaging System a) Rabbit IgG probed with Cy3–tagged goat anti-rabbit IgG b) Mouse IgG probed with Cy5-tagged goat anti-mouse IgG c) Multiplex fluorescent detection of two fold serial dilution of mouse and rabbit serum proteins probed with Cy3–tagged goat anti-rabbit IgG and Cy5-tagged goat anti-mouse IgG, respectively, on same immunoblot. CyDyes were imaged using a one second exposure with the BioSpectrum Imaging System. Once acquired, the original unaltered image was archived and a copy was used for image analysis. The Fluorescent western blotting applications offer many image was adjusted to globally remove background advantages over chemiluminescent or chromogenic intensity and contrast, and was pseudocolored green visualization. Most significantly, fluorescent labels and red using VisionWorksLS Software to indicate Cy3 permit multiplexing so that several proteins in a sample and Cy5, respectively. can be detected and analyzed at the same time and on a single protein blot. Fluorescent labels in particular offer very low background and a high signal-to-noise ratio for quantitative imaging. Dye Excitation Emission Cy3 525BP45 605BP50 The combination of the BioSpectrum Imaging System Cy5 630BP30 730BP40 and the BioLite MultiSpectral Light Source provides not only a full range of wavelengths for excitation light but Table 1. Filters used for NIR blotting with 680 and 770 also rapid high resolution image capture through the use to 800nm fluorescent tags. of deeply cooled MegaCam 810 and OptiChemi™ 610 cameras and low light lenses. Results and Discussion Imaging Time Figure 2 illustrates the multiplex imaging capabilities of the BioSpectrum Imaging System, specifically Compared to laser scanning imaging systems where separating out the signal of both Cy3 and Cy5 color imaging times range from 3 to 5 minutes, the channels. BioSpectrum Imaging System provides a significant advantage with imaging times ranging from 1 to 5 seconds for fluorescent Western blotting applications.
  • 3. Focal Points Application Note Page | 3 Conclusion References Fluorescent Western blot imaging with the BioSpectrum 1. Gallagher, S.R. and Wiley, E.A. Current Imaging System and BioLite MultiSpectral Light Source Protocols: Essential Laboratory Techniques. is a fast and efficient process for enabling researchers Wiley, 2012 to achieve simultaneous detection of multiple proteins on the same immunoblot and generate high resolution, publication-ready images. Figure 3. BioSpectrum Connected to the BioLite Together, the BioSpectrum and BioLite are a powerful combination that are able to specifically excite and illuminate at wavelengths from 365 to 765nm and read emissions from 400 to 850nm. Use of up to eight excitation and five emission wavelengths are possible in a single experiment. The BioSpectrum 810 and BioSpectrum 610 are recommended for imaging multiplex fluorescent Western blots.
  • 4. Focal Points Application Note Page | 4 BioSpectrum and VisionWorks are registered trademarks of UVP, LLC. BioLite and OptiChemi are trademarks of UVP, LLC. All other registered trademarks or trademarks are the property of their respective owners.