Scanning and Analyzing Western Blots Using LiCor Odyssey

Materials

• LiCor Odyssey (with rubber mat)
• Dry Western Blot
• Roller
• Plastic forceps (attached to Odyssey)
• DI water
• KimWipes
• USB (for data and pictures)

Getting Started

1. Clean glass scanning surface of the Odyssey gently with DI water and KimWipes to remove any dust or debris.
2. Open ImageStudio (yellow icon on desktop). Click on or create (using your last name) your Work Area.

Image Aqusition Protocol

1. Place blot (wet membrane if scanning for total protein, dry membrane if scanning finished blot) on scanner, protein side down. Cover with clear rubber mat and roll with roller to remove any air pockets. Take mental note of where your blot is on the ruler on the scanning surface.
2. With the 'Acquire tab selected, click on the blue box in the large white-grid working space and drag edges to fit around your blot. (Tip: Note the coordinates of your blot using the ruler-as above-and set blue box wider that blot initially).
3. Click Preview on the top right menu bar (blue circle with magnifying glass) to scan a preliminary, low resolution image. Use this image to capture all of the blot. Adjust and re-scan with preview as needed. After the scan is complete, a pop-up menu with come up to adjust the image. Click Cancel.
4. Add specifics to the image table (antibody, experiment, name of file: yy-mm-dd_nameofexperiment).
5. Adjust the blue box to fit just around your blot.
6. Re-scan for a high-resolution image by cliking Start on the top right menu bar (green circle with power button icon). Press Cancel on the image-adjuster pop-up.

Analysis/Quantification

1. With the Image tab selected, select the image you want to analyze from the table at the bottom.
2. You can flip, rotate, and straighten the image to better align the lanes using Rotate or Flip' and Free Rotate in the top center menu. Using these tools will automatically create a new, adjusted image with a description of the changes from the original in the Images table.
3. With the Analysis tab selected, Select Western"' as a Type on the leftmost side of the menu bar. This will create a second Analysis tab.
4. Adjust the Number of Lanes on the far left of the menu bar to fit your blot.
5. Click Redraw Boundaries and drab the boundary to include all lanes (not ladder(s)).
6. To select the bands of interest, select the 700 or 800 channel by clicking the X box next to the channel on the right side of the screen (with the vertical Display tab selected). Add boxes to your bands (see below) and repeat for each channel. Each band of interest should have one box around it.
   1. You can use Add in the Bands menu in the top center menu bar to add boxes around individual bands to be quantified.
   2. Or you can use Add to All Lanes in the Bands menu to add bands of one size across all lanes. This is much faster if you have many lanes.
7. Click Select in the Bands menu.
8. Click the vertical Profiles tab (below the Display tab. Adjust the edges of the boxes around each band to include the entire band using the graphs. Each edge should be on the axis and should capture the desired peak(s).
9. Once all bands are adjusted, click the Western Bands tab within the table at the bottom of the screen.
10. Click the triangle in the top left corner of the table to copy the table (or the lanes you want). Copy into an Excel spreadsheet or a Text file to use in Excel on your own computer.
    1. Use this format: yy_mm_dd_nameofexperiment_quantification
11. Save your work/Work Area within ImageStudio and save pictures (without features) to your flash drive. You can remove the features by unchecking the boxes in the Show menu bar on the right of the top menu bar.
    1. For the former, click the disk icon at the top the screen.
    2. For the latter, click the yellow IS circle at the top left of the screen and select Export and Image for Print. Save as TIFF, PNG, or PDF as needed. (Note: Save pictures in greyscale by clicking the greyscale box in the Display tab above Profiles (see #6).
    3. You can also save everything in to a Lab Notebook as a PDF using the Lab Book tab in the top menu.

Note: To quantify the total protein scan, use steps 4-7, but with adjustments to 7.

1. Once the boundary is set, click Add or Add to All Lanes to each of your bands of interest. You'll only do this in the 700 channel, as the total protein stain is only for the 700 channel.
2. Control+Click to select all bands in one lane and click Merge in the Bands menu to create one box around the lane.
3. Adjust the edges, as above.
4. Use the Western Bands' tab, as above to copy the quantification values (see #9).

Finishing Up

1. Check that you have collected and saved all data and pictures to your Work Area and to your flash drive (later transfer to the shared drive).
2. Either close our your Work Area or close the program.

• For the former, click the yellow IS--> Work Area-->Switch.

1. Collect blots and save in tin foil with notes in your lab notebook.