Image Acquisition

Using Membranes

For Western blotting methods, nitrocellulose or PVDF membranes may be used (see licorbio.com/membranes for the latest membranes and kits that are optimal for infrared detection). Detailed blotting protocols can be found at licorbio.com/support. These are some general tips for using membranes.

Handling Near-Infrared Western Blot Membranes

  • Important: Do not touch the membrane – handle only with clean forceps. Lift the membrane only by the corners. Fingerprints, even from a glove, will show clearly when imaged.

  • Use clean containers to avoid cross-contamination and reduce background.

  • Protect near-infrared (NIR) Western blot membranes from light prior to imaging.

  • Keep the membrane wet if it is to be stripped and re-used.

  • Store in PBS or TBS at 4 °C or store dry at room temperature. The fluorescent signal on a dried membrane will remain stable for several months or longer if protected from light and air.

Acquiring NIR Western Blot Images

NIR Western blot membranes can be imaged wet or dry. Place the membrane sample-side up in the imaging tray, and place the imaging tray into the imaging drawer.

LI‑COR Acquisition Software Steps for Scanning Membranes

  1. Start LI‑COR Acquisition Software LI-COR Acquisition icon.

  2. Click Scan LI-COR Acquisition scan icon.

  3. Enter a Username.

  4. Choose your Odyssey® XF in the list of imagers.

  5. Choose the Membrane workflow and follow the steps in the workflow to acquire an image.

Handling Chemiluminescent Western Blot Membranes

  • Important: Do not touch the membrane – handle only with clean forceps. Lift the membrane only by the corners. Fingerprints, even from a glove, will show clearly when imaged.

  • Use clean containers to avoid cross-contamination and reduce background.

  • Do not dry chemiluminescent membranes for imaging.

Acquiring Chemiluminescent Western Blot Images

Image chemiluminescent membranes wet and with enough substrate evenly dispersed.

LI‑COR Acquisition Software Steps for Scanning Membranes

  1. Start LI‑COR Acquisition Software .

  2. Click Scan LI-COR Acquisition scan icon.

  3. Enter a Username.

  4. Choose your Odyssey XF in the list of imagers.

  5. Choose the Membrane workflow and follow the steps in the workflow to acquire an image.

    The following imaging settings are used in this workflow. Other settings can be entered in the Custom workflow.

    • Chemi Acquisition Time Options: 30 sec, 1 min, 2 min (default), 5 min, 10 min, 20 min, 60 min

    • Chemi Manual Entry for Acquisition Time: 5 seconds to 60 minutes

Using Gels

DNA gels that are stained with ethidium bromide or other comparable stains (such as SYBR® Safe) can be imaged in the 600 nm channel. A detailed technical note on imaging DNA gels can be found at licorbio.com/support. Coomassie-stained gels can also be imaged since Coomassie Blue dye can be seen clearly in the 700 nm channel. To image a gel, observe the following guidelines:

  • Thoroughly rinse the gel with destaining solution or water to remove dye particulates.
  • When placing the gel in the imaging tray, place the top toward the back of the instrument.

LI‑COR Acquisition Software Steps for Scanning Membranes

  1. Start LI‑COR Acquisition Software LI-COR Acquisition icon.

  2. Click Scan LI-COR Acquisition scan icon.

  3. Enter a Username.

  4. Choose your Odyssey XF in the list of imagers.

  5. Choose the Gel workflow and follow the steps in the workflow to acquire an image.

    The following imaging settings are used in this workflow. Other settings can be entered in the Custom workflow.

    • Resolution: 125 µm

    • Fluorescence Acquisition Time Options: 30 sec, 1 min, 2 min (default), 5 min, 10 min