TITLE: Western Blot Detection using Licor NIR Fluorescence - CHEM 584 
AUTHORS: Ken Christensen

[STEPS]
?. [After the transfer, complete the following steps]
Wet in 1x PBS or TBS for , or until fully hydrated (using the appropriate buffer system).
?. [After the transfer, complete the following steps]
Place membrane in incubation box and block the membrane in 5% milk (PBS or TBS +0.2% v/v Tween® 20) for  with gentle shaking. Be sure to use a sufficient blocking buffer to cover the membrane (a minimum of 0.4 mL/cm2 is suggested).
?. [After the transfer, complete the following steps]
Prepare primary antibody:a. Dilute primary antibody in Odyssey Blocking Buffer + 0.2% Tween 20, using the vendor’s recommended dilution for Western blot applications. Dilutions typically range from 1:200 - 1:5,000, depending on the primary antibody.b.Use enough antibody solution to completely cover the membrane. Put the membrane in a small ziplock baggy, this will allow a small volume of antibody to effectively cover the membrane area.
?. [After the transfer, complete the following steps]
Incubate blot in diluted primary antibody for  to * at room temperature with gentle shaking, or overnight at 4 °C. If the procedure cannot be completed in full, this is a good place to stop until the following day. Incubate the blot in primary antibody overnight at 4°C with gentle shaking, and resume the protocol the next day.*NOTE: Optimal incubation times vary for different primary antibodies.
?. [After the transfer, complete the following steps]
After the primary antibody incubation, wash membranes:a. Pour off the primary antibody solution.b. Rinse membrane with appropriate buffer – 1x TBS-T (0.1% Tween® 20) or 1x PBS-T (0.1% Tween 20).c. Cover blot with 1x TBS-T (0.1% Tween 20) or 1x PBS-T (0.1% Tween 20).d. Shake vigorously on a platform shaker at room temperature for .e. Pour off wash solution.f. Repeat additional times.b. Rinse membrane with appropriate buffer – 1x TBS-T (0.1% Tween® 20) or 1x PBS-T (0.1% Tween 20). Do not add SDS.NOTE: A Suggested dilution range for secondary antibodies is typically 1:5,000 to 1:25,000. Recommended dilutions can be found on the secondary antibody pack insert. Use 1:20,000 as a suggested starting point.
?. [After the transfer, complete the following steps]
Protect the membrane from light during incubation. Incubate blot in diluted secondary antibody for  at room temperature with gentle shaking. Protect the membrane from light during washes.
?. [After the transfer, complete the following steps]
Wash membranes:a. Pour off secondary antibody solution.b. Rinse membrane with the appropriate buffer – 1x TBS-T (0.1% Tween 20) or 1x PBS-T (0.1% Tween 20).c. Cover blot with 1x TBS-T (0.1% Tween 20) or 1x PBS-T (0.1% Tween 20).d. Shake vigorously on platform shaker at room temperature for .e. Pour off wash solution.f. Repeat additional times.
?. [After the transfer, complete the following steps]
Rinse membrane with 1x TBS/PBS (as appropriate) to remove residual Tween 20.
?. [After the transfer, complete the following steps]
The Western blot is now ready to image. The membrane can be stored in 1x TBS or 1x PBS for up to 48 hours in the dark at 4°C. If the blot is prepared more than an hour in advance, air-dry the blot and store in the dark at room temperature until ready to image.
?. [After the transfer, complete the following steps]
10. Image with an Odyssey® Family Imaging System. If the signal on the membrane is too strong or too weak, adjust the imaging parameters to optimize the image.