The overall goal of this procedure is to demonstrate a capillary electrophoresis immunoassay using a commercial platform. This platform will distinguish and quantitate protein targets from a biological sample which can be sourced from cell culture tissues and biofluids. The process examines protein based on size that range from two to 440 kilodaltons.
The advantage of this automated procedure over traditional procedures, like a Western blot, is that capillary electrophoretic immunoassays eliminates the need for gels, transfer devices, and manual washes. Also the absolute amount of protein required is approximately 10 times less, making the procedure ideal for rare cell types or limited samples. In addition, results are obtained in as little as three hours using automated systems and have been shown to be more quantitative and reproducible than conventional Western blot procedures.
Prepare the samples and reagents from the kit, including sample buffer, dithiothreitol, fluorescent master mix, and biotinylated ladder according to the manufacturer's product insert. Prepare the protein samples using your favorite method. Here we isolated whole cell extract from BEAS-2B cell cultures.
Dilute the protein samples with sample buffer. Mix one part five-X fluorescent master mix with four parts diluted sample to achieve the desired final protein concentration. An example calculation is shown for making 70 microliters of 0.2 microgram per microliter final protein concentration starting with a protein stock concentration of 1.2 micrograms per microliter.
Master mix is 1/5 of the total volume, in this case, 14 microliters. To calculate the volume of protein stock needed, multiply the desired final concentration by the total volume and divide by the protein stock concentration. Subtract the master mix and stock volumes from the total volume to calculate the volume of sample buffer.
Denature the prepared samples and ladder by heating at 95 degrees centigrade for five minutes. Note, some proteins may require gentler denaturing conditions. For example, 70 degrees for 10 minutes to prevent protein aggregation and improve migration.
Consider this option if there is heavy smearing at the higher molecular weights. Prepare the desired antibody dilutions in the provided diluent. Note, antibodies are generally used at higher concentrations for capillary-based immunoassay than for traditional Western blotting.
Prepare a one-to-one mixture of luminol and peroxide fresh with each use. Pipette the samples and reagents into the assay plate using the volumes shown in the template. Color coding represents the proper loading of reagents and samples to the assay plate.
Pipette biotinylated ladder to well A1, prepared samples to wells A2 through A25. The provided antibody diluent to wells B1 through B25 and well C1.Primary antibody to wells C2 through C25. Streptavidin HRP to well D1.Secondary antibody to wells D2 through D25.
And luminol-peroxide mix to wells E1 through E25. Add wash buffer to the first three rows of the larger mid-plate wells. Turn on the capillary immunoassay instrument and open the accompanying software.
Load the capillary cartridge and the assay plate into the machine according to the manufacturer's instructions and start the run. When the run is complete, check that the fluorescent standards are correctly identified and correct if necessary. In addition, verify that the biotinylated ladder shows the correct sizing peaks for the kit used.
See the manufacturer's quick reference guide or product manual for more details. Optimization of assay conditions such as exposure time, protein concentration, and antibody dilution are important for obtaining accurate, reproducible results. These conditions are specific to each model system antibody combination and therefore, should be determined empirically for each new assay.
The ability to generate quantitative results depends on analysis of an exposure time at which the luminol substrate is not being rapidly depleted, as substrate depletion results in loss of signal or signal burnout. This can be determined by examining the data at different chemiluminescence exposure times as shown in figure two. Exposures with the instrument used ranged from five to 480 seconds.
Lane views showed decreasing protein concentrations for BEAS-2B extracts probed with p53 DO-1 antibody at a one to 500 dilution. Chemiluminescence signal coefficients reported as peak heights in the instrument software are superimposed. Visual bands intensities are automatically adjusted by the instrument and are not comparable from one panel to another.
The signal coefficient decreases with sequentially longer exposures if luminol becomes depleted, as seen here. The signal begins to disappear and the peak splits at the two longest exposures. Because of this, a short exposure time of 15 seconds was chosen for data analysis.
Total protein input should also be optimized for each particular assay and model system. For quantitative assessment, measurements must be taken within the linear dynamic range of each assay. Where changes in signal, as measured by peak area, are proportional to changes in the amount of protein in the sample.
Lysate titration shows the lane views of BEAS-2B lysate when probed with one to 500 p53 DO-1 or one to 50 alpha-Tubulin antibody. Linear regression analysis confirms the assays are linear over the entire range tested. Total protein concentrations in the middle of the linear range were chosen to accommodate potential target protein variation in either direction.
As a final optimization consideration, proper primary antibody dilution should be assessed. Using antibodies at suturating concentrations helps ensure that any signal changes measured are due only to changes in protein amount. Two BEAS-2B protein samples, represented by the blue and orange dots, were probed with serially diluted alpha-Tubulin antibody.
Chemiluminescence signal, measured as peak area, was plotted against antibody dilution. Saturation was observed near the one to 50 dilution, where the curve begins a noticeable plateau. One to 50 was therefore chosen as the optimal dilution for this antibody.
After watching this video, you should feel comfortable preparing samples, loading samples, and executing analysis on the ProteinSimple Wes capillary-based immunoassay instrument. Once you have mastered this procedure, the entire run can be performed in three hours with 25 samples. When analyzing a run, it is important to perform quality control for each capillary and sample.
This includes verifying fluorescent standards in a biotinylated ladder. If peaks are incorrectly identified, the analysis software should be used to mainly identify correct peaks and eliminate incorrectly identified peaks. As with all molecular biology techniques in the laboratory, wear proper personal protective equipment to protect yourself and your samples.
This includes lab coat, gloves, safety goggles, and closed-toed shoes. It is important to keep in mind that optimization should be performed for each new target that is measured or when different sample sources are used. Validation and follow-up steps include assessing antibody specificity and signal using purified protein or epitope.
Testing the dynamic range of the assay using a series of the sample dilutions and optimizing antibody dilution by assessing signal saturation over an antibody concentration range. Once optimized, this capillary immuno procedure should provide more rapid, more quantitative method of measuring target protein of biological samples compared to traditional methods such as Western blot.
