15.6: Exercise 2 - Immunodetection

This is a multi-day procedure. Timing may vary for different classes. Membranes are rehydrated and treated with blocking reagents

1. Wearing GLOVES, unwrap the dry blot from the plastic wrap. Use the prestained standards to identify the side of the membrane to which the proteins are bound. Submerge the membrane in methanol with this side facing up. Gently agitate the membrane by hand rocking for 30-60 seconds until the membrane has been uniformly wet with methanol. Decant the methanol into the appropriate container and fill the tray half way full with deionized water. Gently agitate the membrane for an additional minute.
2. Decant the water and replace it with sufficient TBS-T (Tris buffered-saline containing 0.05% Tween 20) to cover the blot. Place the blot on a rocking platform. Equilibrate the blot in TBS-T for 5 minutes with slow rocking. At the end of 5 minutes, drain off the TBS-T.
3. Pour enough blocking solution (5% nonfat milk in TBS-T) onto the blot to cover it.
4. Cover the tray with a small piece of plastic wrap. Label the tray clearly and place the tray on a rocking platform in the cold room. The blot should float freely in the tray so that both sides are washed. Incubate the blot for at least an hour or up to 24 hours at 4 ̊C.

Membranes are washed and incubated with primary antibody (~24 hours)

1. Locate your blot in the cold room and bring it back to the lab room.
2. Remove the plastic wrap from the container holding the blot and pour off the blocking solution. SAVE the plastic wrap! You will need it to cover the container again!
3. Add enough TBS-T to cover the blot and place the container on the rocking platform. Rock for 5 minutes.
4. Pour off the TBS-T. Add 15 mL of primary antibody diluted in blocking buffer.
5. Cover the container with the same piece of plastic wrap and place the tray on the rocking platform in the 4 ̊C cold room. Make sure that the blot floats freely in the tray and that the standards are on the top face of the blot. Incubate overnight at 4 ̊C with slow rocking. NOTE: The timing of this step is the most critical in the procedure. Shortening the incubation time with primary antibody may reduce the sensitivity of the western blot.

Secondary antibody binding and detection (1.5-2 hours)

1. Locate your blot in the cold room and bring it to your lab classroom.
2. Carefully drain the antibody from the blot into the test tube marked “Used primary antibody”. (Antibodies are expensive. Fortunately, the solutions can be re-used.)
3. Fill the tray with the blot about half-full with TBS-T. Place the tray on a rocking platform and wash the membrane for 5 minutes to remove unbound primary antibody. Drain the TBS-T when the wash is complete.
4. Repeat step 3 once more, for a total of two washes.
5. Add enough secondary antibody to cover the blot and incubate the membrane for 1 hour with gentle rocking at room temperature. The secondary antibody, which is conjuated to horseradish peroxidase (HRP), has been diluted in TBS-T.
6. Carefully drain the antibody from the blot into the test tube marked “Used secondary antibody.”
7. Wash the membrane 3 times for 5 minutes each with TBS-T, as in step 3.
8. Drain the TBS-T from the blot. Using a P1000 micropipette, cover the blot with 1 mL of 3,3’5,5’-tetramethyl benzidine (TMB), a colorigenic substrate for HRP. Let the color continue to develop until distinct bands are apparent. Bands will probably become apparent within minutes. Do not allow the blot to over-develop, when nonspecific bands become apparent.

9. Stop color development by diluting the substrate with an excess of deionized water. Drain the diluted substrate into the waste container.

10. Allow the blot to dry on a piece of filter paper. Record your data with your cell phone camera.