A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

After collecting 100 microliters of human platelet lysates from ALS patients and healthy subjects, fill out in-house generated templates for the capillary layout and sample preparation. The sample mixture preparation table is dynamic and will automatically calculate how much volume should be removed from the source. Place all the assay reagents on ice, except the standard pack which should remain at room temperature.

To prepare the fluorescent master mix, add 40 microliters of deionized water to a clear tube of DTT, and add 20 microliters of 10X sample buffer, and 20 microliters of the prepared 400-millimolar DTT solution to the pink tube from the assay kit.

To prepare the biotinylated ladder, add 16 microliters of deionized water, 2 microliters of 10X sample buffer, and 2 microliters of the prepared 400-millimolar DTT solution to the white tube provided in the kit. After gentle mixing, transfer the ladder solution into a 200-microliter PCR tube before denaturing.

Add 1.5 microliters of 10X sample buffer and 148.5 microliters of deionized water to a 600-microliter microcentrifuge tube. and vortex to mix, before placing the tube on ice. Add the antibodies of interest directly to the diluent, as indicated in the table, and flush the pipette tip multiple times to obtain a homogeneous antibody solution.

To prepare the capillary sample mix, open all of the PCR tubes, and add 1.6 microliter aliquots of fluorescent 5X sample buffer to each tube, as indicated in the table, using a reverse pipetting technique, immediately closing each tube as the buffer is added. When all of the buffer has been added, open all of the tubes, and add a 0.1x sample buffer in volumes as indicated in the table, immediately closing each tube as the buffer is added.

Next, open all of the tubes, and add the protein samples as indicated in the table, and immediately close each tube cap. When all of the samples have been added, briefly centrifuge all of the tubes in a benchtop centrifuge. Vortex to mix, and centrifuge the samples again.

At the end of the second centrifugation, place all of the tubes into a thermocycler with a heated lid, and denature the samples under the indicated conditions. At the end of the denaturation, centrifuge, mix, and centrifuge the samples again, and place all the tubes in a tube rack on ice.

Using the figure as a guide, add 10 microliters of streptavidin horseradish peroxidase to well D1, 10 microliters of the appropriate secondary antibody to wells D2 through D5, 10 microliters of antibody diluent to each well in row B and to well C1, 10 microliters of the appropriate primary antibody to wells C2 through C25, 5 microliters of biotinylated ladder from PCR tube number 1 to well A1, 3 microliters of sample to rows A2 through A25, and 15 microliters of freshly prepared luminol peroxide to each well of row E.

Pipetting the sample mix and the reagents into the tiny well in the correct orders is critical to the success of experiment, so it should be done very carefully.

Then add 500 microliters of wash buffer to each designated wash buffer well, and spin down the plate contents by centrifugation.