Detection Sensitivity of Carestream Image Station

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ECL Plex Fluorescent Rainbow Marker (GE. Healthcare ... XP marker (Invitrogen, Carlsbad, CA, USA). ... with mouse anti-ß-actin (Sigma Aldrich, ST. LOUIS,.
AP0075 Mar 2011

Detection Sensitivity of Carestream Image Station 4000MM PRO for Three Commonly Used Fluorescent Dyes of the CyDye Family (Cy3, Cy5 and Cy7) M. Catherine Muenker and John Pizzonia Author Information: Molecular Imaging, 4 Research Drive, Woodbridge, CT 06525 Key Words: Multiplexing, Western blot, Fluorescent CyDyes, PVDF membrane, Protein Detection

Introduction Fluorescent dyes with longer wavelength emission maxima in the visible and near infrared ranges have become valuable tools in Western blotting applications. In particular the sulfonated indocyanine fluorescent dyes, CyDyesTM, (GE Healthcare, Piscataway, N.J., USA) are a single family of small bright fluorescent molecules that are widely used for labeling of biomolecules including antibodies and other proteins1,2. These molecules all derive from a common core structure (see Figure 1, top). Among their desirable properties are good aqueous solubility and relative insensitivity to pH changes between 3 and 10. They exhibit low non-specific binding permitting reliable activity in a wide range of buffers. CyDyes also have good photostability, high molar extinction coefficients and favorable quantum yields3. They are available in a variety of chemistries making them suitable for labeling via amine, thiol, and aldehyde groups4,5,6. The purpose of the following study was to determine the detection sensitivity of the Carestream Image Station 4000MM PRO (IS4000MM PRO) with 10 X zoom lens for three commonly used members of the CyDye family (Cy3, Cy5 and Cy7) and to demonstrate their usefulness in multiplex Western blotting experiments. The 3 dyes have non-overlapping emission spectra (see Figure 1, bottom) making them ideal for the design of multiplexing protocols. They are routinely conjugated to IgG antibodies with approximately 6 to 9 moles of dye/antibody ratios. While specific labeling information is not

Figure 1. Chemical structure (top) and excitation and emission spectra (bottom) for Cy3, C C

available from all suppliers, higher ratios have been reported by Berlier and co-workers1 that result in selfquenching and diminished signal output. Quantum yields for these three forms of the fluorochromes have been estimated as > 0.15 for Cy3, > 0.28 for Cy5, and > 0.25 for Cy7.

Methods Slot Blot Preparation For this analysis, stock reagents of approximately 1 mg/ml Cy3, Cy5, and Cy7 conjugated secondary antibodies were diluted 1:50, 1:500, and 1:5000 in phosphate buffer solution [PBS], and than serially diluted 1:2 to yield 8 steps. A Schleicher and Schuell Minifold II Slot Blotter was prepared with two pieces of Whatman 3 mm filter paper and a rehydrated piece of Immobilon™-FL (GE Healthcare, Piscataway, NJ, USA) polyvinylidene fluoride (PVDF). This membrane was chosen due to the extremely low autofluorescent background particularly in the green emitting region where Cy3 emission occurs. Rehydration was accomplished by placing the membrane into methanol for 30 sec, water for 1 min, and then into PBS before being placed into the slot blotter. A 75 µL aliquot of 5% sucrose was pipetted into each chamber slot as a density gradient to prevent leaching of sample onto the PVDF prior to vacuum suction and also to prevent membrane drying during the subsequent pipetting of additional samples. The Cy dyes samples were pipetted into the slot blotter in 50 μL aliquots, the apparatus was vacuumed briefly until the wells were empty, and the membrane was immediately immersed in PBS to prevent drying. Blots were placed onto the platen of an IS4000MM PRO, covered in PBS, and imaged using the protocol described below.

Figure 2. Excitation and emission filters used on the IS4000MM PRO for imaging Cy3 (top), Cy5 (middle), and Cy7 (bottom).

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Cy7 slot blots were imaged using the excitation and emission filtering strategies shown in Figure 2. For image acquisition of Cy3, a 510nm excitation filter and a 570nm emission filter were paired. For Cy5 a 630nm excitation filter and a 670nm emission filter were paired, and for Cy7 a 730nm excitation filter and a 790nm emission filter were paired. Individual parameters for image acquisition are listed in the file history presented with each Figure. All images taken were lens corrected prior to data analysis.

Western Blot Preparation A multiplex Western blot using Cy3- and Cy5labeled secondary antibodies (GE Healthcare, Piscataway, NJ, USA) was imaged using an IS4000MM PRO with a 10X zoom lens (Figure 6). The Western blot was prepared by loading a 4-12% polyacrylamide gel with a lane of Cy3/Cy5 ECL Plex Fluorescent Rainbow Marker (GE Healthcare, Piscataway, NJ, USA), 4 lanes of 10 µg NIH/3T3 cell lysate (Rockland Immunochemicals, Gilbertsville, PA, USA), and a lane of Magicmark XP marker (Invitrogen, Carlsbad, CA, USA). After electrophoresis for 1.5 h at 180V, the gel was transferred onto Immobilon-FL PVDF (Millipore, Billerica, MA) overnight at 30V in a 4˚C refrigerator. The blot was blocked for 30 min with a solution composed of 0.2% casein in PBST (PBS plus 0.01% Tween-20). Next the blot was incubated with mouse anti-ß-actin (Sigma Aldrich, ST. LOUIS, MO) and rabbit anti-ERK1,2 (Abcam, Cambridge, MA) primary antibodies simultaneously in HIKARI solution 1 (Nacalai, USA, San Diego, CA). After the primary incubation the blot was washed 3X in PBST. Finally, the blot was incubated in goat antimouse Cy3 and goat anti-rabbit Cy5 secondary antibodies at 1:2000 in HIKARI solution 2, followed by 3 washes in PBST, then a final PBS wash prior to imaging. . Image Acquisition The IS4000MM PRO, with an automated zoom lens, uses a 400 watt Xenon illuminator to provide a broad range of excitation energy across the visible spectrum. The excitation wavelength is refined using filters that have a 20nm full width at half maximum transmission (FWHM) in 10nm increments from the violet (440 nm) to the near IR (830 nm). The emission filters have a wider profile using a 35 nm FWHM bandpass. Additionally, the emission filters have a patented wide angle coating that reduces stray light artifacts. The Cy3, Cy5 and

The Western blot was imaged with the following image parameters; f-stop at 2.8, FOV at 80 mm, focal plane at 0 (Platen surface), and binning at 2x2, with an autoselected correction file for the optimal filter set of each Cy dye. The Cy3 image was taken for 1 minute using the 510 nm excitation filter paired with the 570nm emission filter and the Cy5 image was taken for 1 minute using the 630nm excitation filter paired with the 700 nm emission filter. The images were tiled in Carestream MI software (v5.0.5), pseudocolored, and overlayed by selecting the Overlay and Transparency buttons in the image display window. The final adjusted image was then exported as an “.emf” file on a Windows® based computer. For use with a Macintosh® based computer, the file can be saved as a “PICT” file. Once opened, the file can be saved as a jpeg or tiff.

Results For the present analysis each blot was imaged twice. The first image was acquired using an exposure time that created the highest signal intensity without saturation (i.e. no pixels exceeding 65,536 grayscale levels). This image is presented in the upper right hand corner of the each Figure and is used to generate the curve defining linearity displayed in the lower right corner of each Figure. Finally a second image was taken to determine the maximum detection sensitivity for each blot. Since this resulted in many of the bands in the first 2 columns being saturated they were omitted from the image presented on the left hand side of each Figure.

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than required for Cy 3 and 4 times longer than was n

Figure 3. Slot blot analysis of Cy3. Upper right – unsaturated image capture (f2.8, 5 sec, no binning), Lower right – demonstration of linearity, Left – Detection sensitivity (f2.8, 60 sec, no binning).

Figure 4. Slot blot analysis of Cy5. Upper right – unsaturated image capture (f2.8, 15 sec, no binning), Lower right – demonstration of linearity, Left – Detection sensitivity (f2.8, 60 sec, no binning).

The data for Cy3 are presented in Figure 3. The first image capture was accomplished using a 5 sec exposure and no binning (upper right). The combined data show excellent linearity (r2 > 0.98) across dilutions (lower right). In this particular experiment the limit of detection was approximately 156 pg of conjugated dye. The data for Cy5 are presented in Figure 4. The first image capture was accomplished using a 15 sec exposure and no binning (upper right). Again the combined data show excellent linearity (r2 > 0.98) across dilutions. While the exposure time to reach sub-threshold saturation was approximately 3 times longer than was observed for Cy3, the ultimate detection sensitivity was still realized with a 60 sec exposure (no binning). In this particular experiment the limit of detection for Cy5 was approximately 156 pg of conjugated dye, similar to Cy3.

Figure 5. Slot blot analysis of Cy7. Upper right – unsaturated image capture (f2.8, 60 sec, no binning), Lower right – demonstration of linearity, Left – Detection sensitivity (f2.8, 10 min, 4x4 binning).

The data for Cy7 are presented in Figure 5. With this dye the first image capture required a 60 sec exposure (no binning, upper right) to reach subthreshold saturation. This is nearly 12 times longer

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IS4000MM PRO when using spectrally unique Cy dye labeled antibodies. In the present example Cy3 and Cy5 were used for Western blotting, but any combination of Cy 3, Cy5, and/or Cy7 would be permissible. The Cy dyes were easily distinguished using the optimal filter sets for each fluorophore. After image capture, the Cy3 and Cy5 images were analyzed, pseudocolored, and overlayed using the MI software for future publication.

than necessary for Cy5. This difference was noted despite the normalization of excitation illumination power across the 3 fluors. Ensuring maximum detection sensitivity required a 10 min exposure using 4 x 4 binning. While the combined data show very good linearity (r2 > 0.97) across dilutions, the detection limit, determined by visual inspection, was slightly higher (~313 pg) compared with Cy3 or Cy5 (~156 pg). Finally, the utility of these reagents for multiplexed Western blotting is presented in Figure 6. In this experiment 2 different proteins were detected from 10 µg of NIH/3T3 cell lysate. The first protein, ERK1,2, is a relatively low abundance protein that separates as 2 bands at 41 and 43 kDa respectively. The second protein is the more highly expressed house keeping gene product ß-actin which separates to about 42 kDa. These very closely associated bands were easily detected using a 60 sec exposure with 2 x 2 binning. Additionally, the utility of using a multiplexed fluorescence approach is underscored by the ease with which the proper identities of each of the bands (ERK1,2, pseudocolored green and ß-actin, pseudocolored red) are reliably confirmed. This experiment illustrates the time-saving ability of conducting a simultaneous western incubation of target and control proteins using spectrally unique fluorescent secondary antibodies.

Conclusion In summary, the data presented support the reliability and detection sensitivity of the IS4000MM PRO for imaging the common cyanine dyes Cy3, Cy5 and Cy7 in applications using membrane formats. Overall, the Cy 3, 5, and 7 dyes showed linearity over 4 orders of magnitude and were sensitive down to 156 pg, 156 pg, and 313 pg, respectively. When conducting sensitivity experiments, careful consideration should be given to choose plates and/or membranes that will contribute the lowest possible background for the wavelengths needed. For these experiments, Immobilon-FL was chosen for having the lowest background fluorescence when compared to other PVDF and nitrocellulose membranes. This experiment also demonstrated that simultaneous or multiplex Western blot analysis is achievable on an

Figure 6. Dual-label Western blot using anti-ß-actin at 42 kDa (Cy3, red pseudocolor) and anti-ERK1,2 at 41 and 43 kDa (Cy5, green pseudocolor) (f2.8, 60s, 2x2 binning). Lane 1 contains Cy3/Cy5 marker, Lanes 2-5 contain 10 µg 3T3 cell lysate, and Lane 6 contains Magicmark XP marker.

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Fradelizi, J., Friederich, E., Beckerle M.C., and Golsteyn R.M., 1999, Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies. Biotechniques 26:484–486. Berlier, J., Rothr, A., Buller, G., Bradford, J., Gray, D., Filanoski, B., Telford, W., Yue, S., Liu, J., Cheung, C-Y., Chang, W., Hirsch, J., Beechem, J., Haugland, R. and Haugland, R., 2003. Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates. J Histochem Cytochem 51:12 1699-1712. Brismar, H., Trepte, O., and Ulfhake, B., 1995, Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, Texas Red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope. J Histochem Cytochem 43:699–707.

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Flanagan, J.H. Jr., Khan, S.H., Menchen, S., Soper, S.A., and Hammer R.P., 1997, Functionalized tricarbocyanine dyes as near-infrared fluorescent probes for biomolecules. Bioconjug Chem 8:751–756. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J/, and Waggoner A.S., 1993, Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem 4:105–111. Toutchkine, A., Nalbant, P., and Hahn, K.M., 2002, Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility. Bioconjug Chem 13:387–391.

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© Carestream Health, Inc., 2011. All rights are reserved. No section of this document may be photocopied, reproduced, translated to another language, stored in a retrieval system, or transmitted in any form without the prior written consent of Carestream Health, Inc. The information contained in this document is subject to change without notice. Carestream Health, Inc. makes no warranty of any kind with regard to this written material. Carestream Health, Inc. assumes no responsibility for any errors that may appear in this document.

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