Multiplex imaging of Q-Dot fluorescent dyes with Vilber Lourmat imaging systems
An application note to show how different Q-Dot® dyes can be imaged and presented as multiplex layer image.
(Martin Auer und Karin Widulle, Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany, email@example.com)
Q-Dot® fluorescent dyes have recently emerged as a powerful tool for detection of blotted proteins on membranes. They exhibit several features that make them unique among the broad range of fluorescent dyes. In contrast to other commonly used dyes, Q-Dot® nanocrystals do not show any bleaching,
they may be excited for as long as 48 hours. Furthermore, due to their broad UV excitation range with optimum below 450 nm and their individually different, narrow emission spectra between 500 and 800 nm, they are highly suited
for multiplex applications providing the possibility to detect several signals in one blot without stripping and re-probing of the membrane.
Nevertheless, as attractive Q-Dot® dyes may be due to their characteristics, it needs to be considered that their excitation as well as their detection requires specially equipped imaging systems. Especially the use of PVDF membranes for Western blotting excludes the use of UV transilluminators, as PVDF is impermeable for UV light. Additionally, PVDF membranes show an extended degree of autofluorescence compared to other materials.
To obtain more insight into the above problematic, Vilber Lourmat has tested two frequently used imaging systems in combination with different camera filters and illumination sources about their ability to detect Q-Dot® fluorescent dyes on PVDF membranes.
An 8% SDS-PAGE gel was prepared according to standard protocol. The twelve lanes were divided into three groups of four lanes each: the first lane containing 15 µl of a CHO total-protein cell lysate as a negative control, the second lane loaded with 5 µl (2.6 µg), the third lane with 10 µl (5.2 µg), the
fourth lane with 15 µl (7.8 µg) of the Broad Range Biotinylated Protein Marker (BioRad 161-0319). SDS-PAGE gel electrophoresis was performed for 90 min at 90 volts. Blotting of the gel onto a PVDF membrane (Hybond-P PVDF, Amersham
Pharmacia) was accomplished by semi-dry method according to standard protocols for 1 hour, before blocking in 3% BSA for 1 hour. The membrane was cut into 3 pieces, lane 1 - 4, lane 5 - 8 and lane 9 - 12, each piece containing identical loading as described before. The first piece of the membrane (lane 1 - 4) was incubated with Q-Dot® 565 streptavidine conjugate (Invitrogen Q10131MP), the second piece of membrane (lane 5 - 8) was incubated with Q-Dot® 655 streptavidine conjugate (Invitrogen Q10121MP), the third piece of membrane (lane 9 - 12) was incubated with Q-Dot® 705 streptavidine conjugate (Invitrogen Q10161MP). For incubation, the Q-Dots® were diluted in PBS according to the manufacturer´s protocol, the membranes were wrapped in foil for protection and then incubated on a shaker overnight at 4°C. Before documentation, the membranes were washed 3 x 10 min in PBS.
The detection of the Q-Dot® signals was performed with the following imaging instruments and accessories:
- Chemi-Smart 3000 combisystem for chemiluminescence and fluorescence; darkroom CN-3000 with 2 x 8 W epi- UV illumination at 365 nm
- Bio-Vision++ fluorescence gel documentation system; darkroom CN-3000 with 2 x 8 W epi-UV illumination at 365 nm
- Starlight module RGB for cold-light LED epi-illumination at 470 nm
- Q-Dot® spectral camera filters: F-Q.565 M58, F-Q.655 M58, F-Q.705 M58
Figure 2) shows the fluorescence images obtained with the Chemi-Smart 3000/2 x 8 W epi-UV illumination at 365 nm. When using the corresponding camera filters, the Q-Dot® 565, 655 and 705 signals are already visible after exposure times of as short as 0,200 sec up to max. 1,100 sec, minimizing the
background autofluorescence signal of the PVDF membrane.
Figure 3) shows the images acquired with the Bio-Vision++,when using 2 x 8 W epi-UV illumination at 365 nm. The Q-Dot® 565, 655 and 705 signals are also clearly visible, the exposuretimes vary between 0,280 sec and 2,120 sec.
Figure 4) shows an example of epi-illumination with the Starlight module (470 nm), integrated in the Chemi-Smart 3000 system. The UV-epi illumination of the darkroom was turned off for this experiment. The figure shows, that Q-Dot® 655 dye is highly efficiently excited, the signal is already visible after 0,100 sec exposure time.
Figure 5) shows the power of the image acquisition software Chemi-Capt and the possibilities of the system for multiplex imaging. After excitation of the signal with the Starlight module, two separate images were acquired with the Chemi-Smart 3000 system by using camera filters F-Q.655 M58 and F-Q.705 M58. The images were superimposed and are displayed here in RGB colors.
The experiments showed that both imaging instruments, the Bio-Vision++ and the Chemi-Smart 3000 are suited to detect Q-Dot® streptavidine conjugates, when the camera is equipped with Q-Dot® spectral filters and the darkroom with 365 nm epi-UV illumination. The Bio-Vision++ may be used if the main focus is on fluorescent agarose gels and if Western Blot imaging is performed only from time to time, as it needs a longer exposure time for acquisition of the image, thus leading in a higher background signal. The Chemi-Smart 3000 may be chosen if Western Blot imaging is performed routinely, if autofluorescence of the membrane is critical or if multiplex images have to be obtained. The Starlight module RGB for cold-light LED epi-illumination at 470 nm has shown to be a powerful tool for the excitation of Q-Dot® fluorescent dyes. The image acquisition required only half of the exposure time compared to epi-UV illumination. With the user softwares for both Bio-Vision++ and Chemi-Smart systems, multiplex images can easily acquired and compared. Up to 5 different images obtained with different filters can be superimposed and be displayed e.g. with RGB colors.