Western Blot for Li-Cor® Scanner

1. Setup and run SDS-Page gel as usual, but when loading gel, use less marker (2 microliters is fine) and, if possible, leave one lane open between the marker and your sample.

2. Wearing gloves, take gel to the Biorad transfer apparatus, put about 100mL transfer buffer (58g Tris Base, 29.5g glycine, 2L MeOH, 37.5ml 10% SDS in H2O for a total of 10 L) into plastic tray. Place sheet of transfer paper into tray, making sure there is enough buffer to cover the paper. Place the paper onto the transfer anode plate.

3. Cut a piece of nitrocellulose to the same size as the gel, wet the nitrocellulose in transfer buffer and then place it over the transfer paper on the anode. Roll any bubbles out from under the paper using a broken pipette.

4. Cut away the stacking portion of the gel and discard, invert the gel into the transfer buffer and lift slowly, the gel should slide off. Place the gel onto the nitrocellulose. If there is sufficient transfer buffer, any bubbles can be removed by lifting one corner of the gel and slowly dropping it, or by rolling with a broken pipette.

5. Wet a second sheet of transfer paper in transfer buffer, then place on top of the gel, rolling bubbles out.

6. Lock cathode in place and set machine to run at 20 Volts for 60 minutes.

7. Discard gel; remove nitrocellulose and place in blocking buffer (1% Fish Gelatin in PBS ) in rocker for 1 hour, or overnight at 4° C.

8. Put nitrocellulose in primary antibody solution and leave on rocker for 60 minutes.

9. Wash 3 times in 20 minutes in western wash (1X PBS, .1% Tween (Sigma) in H2O).

10. Put nitrocellulose in secondary antibody solution and leave on rocker for 60 minutes.

11. Wash 3 times in 20 minutes in western wash, leaving the nitrocellulose in the wash.

12. Place nitrocellulose face down in the middle of the LI-COR® Odyssey™ scanner and place the silicone sheet over the nitrocellulose. Work out any bubbles with the silicone roller. Note the location of the nitrocellulose by the numbers around the edge and close the lid.

13. Open the Odyssey™ software from the desktop, and hit Ctrl-N to create a new project.

14. Under the file menu, click "scan", and login with username "licor" and password "licor".

15. Set the size and location of the image to be scanned by clicking and dragging a red box on the grid to match the location numbers from the top of the scanner.

16. For the first scan, set the resolution to 337 microns, quality to lowest, and run the channel(s) appropriate to your secondary antibody (i.e. 700 nm channel for 680 nm anti mouse).

17. Use the low quality scan to ensure that the nitrocellulose is properly aligned and to adjust the intensity of the channels to give the least background.

18. Run a final scan. For most purposes the 169 nm resolution and medium quality settings are sufficient. Save and print your analysis.

19. Discard the nitrocellulose, or save in western wash for reprobe or later analysis. Clean the silicone sheet with isopropanol, and dry the bed of the scanner with a Kimwipe®

By: Brandon Craven