This protocol can be used to optimize both protein and primary antibody concentrations in a single assay plate, and to perform two assays using a single sample preparation. The main advantage of this technique is that it allows 2 1/2 days of lab work including data analysis, to be completed in 3 1/2 hours. This technique provides a high throughput assay format for developing a blood-based biomarker for ALS and is an ideal method for conducting large clinical trials.
This method can provide insight into target protein profiles during disease prognosis and can facilitate the monitoring of the effects of drugs of interesting clinical trials. 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, two microliters of 10X sample buffer and two 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 the diluent as indicated in the table and flush the pipette tip multiple times in a homogenous 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, five microliters of biotinylated ladder from PCR tube number one to well A1, three 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 reagent 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. To perform the capillary analysis on the assay plate, connect the capillary analyzer to the online system and click Instrument and Connect in the analyzer software.
Select the instrument serial number within the popup menu, and click Connect. Select the Assay tab, and select New Assay, enter the assay parameters and save the file name and location. Then confirm that the blinking blue indicator in the analyzer is a solid blue and remove the capillary cartridge from its packaging.
Remove the protective seal from the assay plate and visually observe the pre-filled wells for air bubbles. Place the assay plate in the plate holder and holding the capillary cartridge at an angle, insert the cartridge into the capillary slot and close the analyzer door. Then click Start in the analyzer software.
The use of a whole platelet lysate mixture in the assay may reduce the signal intensity of the target proteins and contribute to a high background signal. Therefore, in this assay, clear supernatant was used after rupturing the platelets. A linear dynamic range for the platelet cytosol protein concentration was established at 0.2 to 0.8 milligrams per milliliter, and an essay template was adopted so that both the protein concentration and the primary antibody titration were able to be performed in a single assay.
The high dynamic range detection profile delivers a significantly wider dynamic range due to the greater sensitivity of the capillary assay, resulting in a better detection and quantification over a larger sample concentration range. Individual exposure times can also be defined. Using the optimized assay conditions, this analysis of platelet lysate cytosolic fractions obtained from ALS patients using two sets of antibodies allow the identification of disease-specific TDP-43 and its phosphorylated derivative within the samples.
The total TDP-43 could then be quantified using the calibration curve. And the intra and inter-run assay variabilities were tested in pooled human ALS platelet cytosolic fractions. This method allows the use of primary antibodies, facilitating the identification of up to five different target proteins within the same sample in a single assay run.
Not only does this technique substantially reduce the protein analysis time, it requires microliter sample volumes and generates results with a high degree of reproducibility. Some of the chemicals used in this assay and all human samples are considered biohazardous, so appropriate personal protective equipment should always be used when performing these experiments.
