Technical Note Use of E-PAGE™ 48- and 96-Well Gels on the Odyssey® Infrared Imaging Systems Developed for: Odyssey® Classic, and Odyssey CLx Imaging Systems Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model. Published October 2008. Revised October 2011. The most recent version of this Technical Note is posted at http://biosupport.licor.com/support.
Page 2 – Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System TABLE OF CONTENTS Page I. Introduction ..........................................................................................................2 II. Required Materials ...............................................................................................2 III. E-PAGE Gel Electrophoresis ................................................................................3 IV.
Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System – Page 3 Semi-Dry Transfer: • Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, P/N 170-3940) • NuPAGE® Sample Reducing Agent (Invitrogen, P/N NP0009)* • NuPAGE Transfer Buffer (Invitrogen, P/N NP0006) • NuPAGE Antioxidant (Invitrogen, P/N NP0005) • Nitrocellulose/Filter Paper Sandwich (Invitrogen, P/N LC2009); or, Invitrolon™/Filter Paper Sandwich (Invitrogen, P/N LC2007)* • Blot Absorbent Filter Paper, extra thick (Bio-Rad, P/N 170-
Page 4 – Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System 10. At the end of the run, the E-Base will beep and the light will flash red. Press the pwr/prg button to complete the run and stop the beeping/flashing. 11. Remove the gel cassette from the Mother E-Base. 12. Transfer immediately using one of the two procedures outlined following (bands in the gel will diffuse within 40 minutes). IV.
Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System – Page 5 11. While holding the tabs together, pull the stack assembly together through the rollers using one smooth motion until it reaches the Gel Barriers on the right side of the blotting surface. 12. Place the Disposable Sponge on the underside of the iBlot device’s lid, with the metal contact positioned at the upper right corner of the lid. Close the lid and secure the latch. 13. Turn on the iBlot power switch.
Page 6 –Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System 4. Carefully remove the trimmed gel from the cassette and place into a clean container with 200 mL of 1X transfer buffer (enough to fully submerge the entire gel). 5. Place the container on an orbital shaker for 30 minutes, with gentle rotation, to allow the gel to equilibrate. 6. Wet two pieces of extra-thick filter paper, one sheet of nitrocellulose or PVDF membrane and the blotting pad (provided with E-PAGE gels) in 1X transfer buffer.
Use of E-PAGE™ Gels on the Odyssey® Infrared Imaging System – Page 7 VI. Sample Data The following data illustrate the utility of E-PAGE gels for high-throughput, quantitative Western blotting. The first example (figure 1) shows how high-quality IC50 data can be generated from several samples on a single E-PAGE gel. This same experiment would require as many as four mini gels to obtain the same data.
Sample Data (CONTINUED) A. B. C. D. E. Figure 2. A. E-PAGE 96-Well gel loaded with replicate untreated or EGF-treated A431 cell extract samples (alternating rows). After transfer with the Invitrogen iBlot system, blots were probed for total ERK2 and phospho-ERK (shown here in a two-color format with both wavelength channels displayed). B. Single (800 nm) channel image showing mouse anti-ERK2 detected with IRDye® 800CW Goat AntiMouse IgG (LI-COR P/N 926-32210). C.
