Example Protocol

Walk through an example protocol using your MPX™ System.

Primary Antibody Application and Incubation

  1. Prepare primary antibody dilutions in Intercept® T20 Antibody Diluent as recommended by the antibody manufacturer. 200 µL of dilution will be needed for each channel used with antibody.

    Dilutions should be made in a microcentrifuge tube if using a single pipette, or a 96-well plate if using a multi-channel pipette. The numbers in the template represent the MPX channel to which the antibody will be applied (Figure 160).

  2. Remove the blocking buffer if needed.

  3. Using your template as a guide, add primary antibody using either a single or multi-channel pipette. Remove bubbles as they occur and make sure all channels contain liquid.

    Note: Unused channels must still be filled with PBS/TBS during the experiment to prevent wicking between channels.

  4. Incubate the membrane with the primary antibody for at least 1-2 hours at room temperature or overnight at 4 °C with gentle shaking.

 Example alignment grid for the MPX
Figure 160.  

Primary Antibody Wash

  1. Remove the primary antibody solution with an 8- or 12-well multi-channel pipette, a single pipette, or vacuum aspiration.

  2. Hold the MPX at an angle over a sink. Flood each channel 3 times with 1 ml of PBS-T (0.1% Tween® 20) or TBS-T (0.1% Tween 20) using either a multi-channel pipette, a single pipette, or a repeating pipette with a 50 ml dispensing tip ().

  3. Wipe off the top of the MPX with a Kimwipe.

  4. Fill each channel with 160 µL of wash buffer (PBS-T or TBS-T) and incubate at room temperature for 5 minutes.

  5. Remove the wash solution with a multi-channel pipette, single pipette, or vacuum aspiration.

  6. Repeat steps 2-5 two more times.

  7. To prevent cross-contamination, aspirate the final wash carefully and completely before beginning the secondary antibody application.

Secondary Antibody Application and Incubation

Using One Secondary Antibody

Follow these steps if you plan on using a single secondary antibody for the entire membrane:

  1. Ensure that the final wash is completely removed.

  2. Open the MPX; remove the membrane with forceps and wash in PBS-T for 10 minutes at room temperature with shaking.

  3. Prepare secondary antibody dilutions in Intercept® T20 Antibody Diluent as recommended. Approximately 0.04 mL/cm2 of diluted antibody will be needed.

    Start with a 1:20,000 dilution for LI‑COR IRDye® 800CW Secondary Antibody, IRDye 680RD Secondary Antibody, and IRDye 680LT Secondary Antibody.

  4. Decant the wash solution.

  5. Cover with diluted secondary for 1 hour.

Using Multiple Secondary Antibodies

Follow these steps if you plan on using multiple secondary antibodies.

  1. Prepare secondary antibody dilutions in Intercept T20 Antibody Diluent as recommended.

    • Start with a 1:20,000 dilution for LI‑COR IRDye® 800CW Secondary Antibody, IRDye 680RD Secondary Antibody, and IRDye 680LT Secondary Antibody.

    • 200 µL of diluted antibody will be needed for each port that will have antibody applied.

    • Secondary antibody dilutions can be prepared in a 96-well plate prior to pipetting into the MPX wells (Figure 158. ) or in tubes if desired.

  2. Use clean tips and fill all channels according to the instruction in How to Load the MPX Using a Single Pipette or How to Load the MPX Using a Multi-Channel Pipette. Unused channels must still be filled with PBS/TBS during the experiment to prevent wicking between channels.

  3. Incubate for 1-2 hours with shaking.

Secondary Antibody Wash

  1. Remove the solution with an 8- or 12-well multi-channel pipette, a single pipette, or vacuum aspiration.

  2. Hold the MPX at an angle over a sink. Flood each channel 3 times with 1 ml of PBS-T (0.1% Tween® 20) or TBS-T (0.1% Tween 20) using either a multi-channel pipette, a single pipette, or a repeating pipette with a 50 ml dispensing tip ().

  3. Wipe off the top of the MPX with a Kimwipe.

  4. Fill each channel with 160 µL of wash buffer (PBS-T or TBS-T) and incubate at room temperature for 5 minutes.

  5. Remove the wash solution with a multi-channel pipette, single pipette, or vacuum aspiration.

  6. Repeat steps 2-5 two more times.

  7. To prevent cross-contamination, aspirate the final wash carefully and completely.

  8. Remove the membrane from the MPX and place it into a large Western blot box containing PBS-T or TBS-T.

  9. Wash for an additional 10 minutes with shaking.