Simultaneous Chemiluminescent and Fluorescent Detection:

A novel approach for double Western blot analysis of low copy protein targets
John H. Pizzonia*, Mary Catherine Muenker, Jens Waldeck and William E. McLaughlin
Carestream Molecular Imaging, 4 Research Drive, Woodbridge, CT, 06525, USA *john.pizzonia@carestream.com

Abstract
Western blotting remains one of the most utilized applications for the identification of proteins. While labor intensive, it is cost effective and can performed with minimal investment. The detection of multiple proteins (a so called Multiplexing strategy), on a blot usually involves processes such as stripping which will remove protein in a non-uniform manner and ultimately degrade the reliability of the data. The recent proliferation in the availability of fluorescent labels makes designing Multiplexing strategies for Western blotting protocols easier, with the major caveat usually coming from the availability of unique 1antibody species. While fluorescence in general demonstrates a wider linear dynamic range than enzyme driven photonic output, the detection sensitive is not on par with chemiluminescent labels using current detection technologies.

HRP Substrate Comparison

Simultaneous Ab Hybridization

For this experiment 3 identical 1:2 serial dilutions of human transferrin protein were transferred to Immobilon-FL and processed using an HRP label. Pierce SuperSignal substrates West Pico (Panel A), West Dura (Panel B), and West Femto (Panel C) were compared and were found to be linear down to 313 pg, 78 pg, and 39 pg, respectively. Again the linearity across the entire dilution for all substrates was very good (R2 > 0.98). Note that as with the first experiment the final data set has been exported into Excel for creating graphs.

Materials/Methods
Precast Nupage 4-12% gradient gels from Invitrogen. Loaded rat HSC70, Human Transferrin or 3T3 cell lysate protein (all obtained from Abcam) on the gels for separation. Transfer the protein to either Immobilon-FL (from Millipore) or Hybond ECL nitrocellulose (from GE) for subsequent detection. Blocking with either blotto (5% non-fat dry milk) in PBST or 0.2% casein in PBST with or without Signal Enhancer Hikari (from Nacali) for 30 min. 1antibody incubations include rat anti-HSC70 (1:5000), mouse anti-Transferrin (1:500), mouse anti--actin (1:2000) or rabbit anti-(ERK)1,2 (1:1000), performed for 1 hr at room temperature. Blots were next washed 3 times in PBST for 5 min, followed by incubation with the appropriate 2species specific antibodies labeled with either Alexa 647 or Horse Radish Peroxidase for 1 hr at room temperature. Finally, the blots were washed 3 times in PBST for 5 min and once in PBS without tween prior to imaging.

Hyb Solution Optimization

This final pilot experiment puts this all together. The blot sequence along the top shows simultaneously incubation of 1 antibodies mouse anti--actin and rat anti-HSC70, followed by simultaneously incubation of 2 antibodies goat anti-mouse HRP, goat anti-rat Alexa 647. For imaging a fluorescence exposure of Alexa 647 was captured first (panel A), followed by addition of the West Femto substrate and chemi imaging (panel B). Finally a second fluorescence image was captured without moving the blot (panel C) and the last 2 images were overlaid as shown in panel D. A graph of the final 3 pilot experiments shown at the bottom confirms that comparable signal intensity can be achieved regardless of the sequence of hybridizations. Note that in this example the data has been exported to Prism for subsequent formatting and presentation.

Image Overlay & Analysis

Results Membrane Selection

Since the goal of this project was to identify a common set of hybridization conditions to perform both chemi and fluorescence reactions, the experiment shown on the left of this figure was performed to look at the effect of blocking buffers and signal enhancer. For this experiment 4 identical 1:2 serial dilutions of rat HSC70 protein were produced, and as can be seen the casein blocker delivered better detection sensitivity when compared with traditional blotto (5% nonfat dry milk). Hikari signal enhancer, combined with casein blocker pushed detection sensitivity resulting in a 8X increase in Alexa 647 detection and a 16X increase for chemiluminescent detection. Signal linearity presented in the Net Intensity plot on the right again shows good r2 values for all plots

Sequential Ab Hybridization

After performing the hybridization and imaging as described in the previous figures the resulting image shown on the left was obtained. As you can see the Alexa 647 2 antibody was used to target the control HSC70 protein and a second HRP-labeled 2 antibody was used to target the low copy ERK 1,2 protein. The results obtained, which is summarized on the right, underscore both the consistency and linearity of chemiluminescence and fluorescence signal regardless of the order in which the images are obtained. It also shows the advantage that chemiluminescence has over fluorescence in terms of S/N, confirming once again the strength of chemi as a label for Western blotting applications. Finally, the images were analyzed, pseudocolored and overlayed with the Carestream MI software and the resulting data exported to Prism to generate the graphs shown on the right.

Conclusion
Sequential incubations of antibodies can be performed without stripping blots. Simultaneous incubations of all 1 and then all 2reagents can be performed provided the species requirements are met. This novel approach to Multiplex Western blotting saves both time and money while providing the same quality and sensitivity as traditionally used Western blotting methods.
For the sequence of images in the top row, the blot was first probed for rat anti-HSC70 using an Alexa label and then imaged (panel A). Next the blot was reblocked and probed with mouse anti--actin antibody using an HRP label (panel B). After the chemi imaging the blot was reimaged for Alexa 647 which still shows up very nicely (panel C). Finally, the 2nd and 3rd images were overlaid as shown in panel D. The order was reversed in blot images shown along the bottom row with the HRP reaction performed and imaged first, followed by reblocking, processing and imaging for Alexa 647. Again both labels are easily detectable.

In this first experiment shown on the left, two identical 1:2 serial dilutions of rat HSC70 protein were transferred onto Immobilon-FL in the upper panel or nitrocellulose (NC) in the lower panel, and chemiluminescent detection was compared. The Immobilon-FL membrane was already shown to be superior for fluorescence detection as it siginficantly reduces background created by excitation light thus improving S/N. For this experiment 3 additional bands were detected on the Immobilon membrane for almost a full order of magnitude greater sensitivity. The Net Intensity data generated on the Immobilon membrane also demonstrated slightly better linearity with an R2 value of 0.98 versus 0.97 for nitrocellulose.

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