5C), and the remaining uncaptured proteins were continuously moving forward. Our results demonstrate that the technique has a broad potential for rapid and cost-effective isotachphoretic analysis of multiplex protein biomarkers in serum samples B-Raf IN 1 at the point of care. and molecular mass of the protein . Under our previous cationic ITP conditions (pH of LE=8, pH of TE =7.2) , proteins with p 7 (such as troponin I) can be electrophoretically stacked, while acidic proteins, p 7 (such as albumin), will be immobile or migrate toward the opposite B-Raf IN 1 direction. To alter the electrophoretic mobility of acidic proteins, antibodies specific to these target proteins can be used. It is known that a typical antibody has a relatively large molecular mass (~ 150 kDa) and basic isoelectric point (p~ 9.0) . It is expected that the immunobinding of a basic antibody and an acidic protein can significantly alter the electrophoretic mobility of the protein. To test this hypothesis, the current study employs bovine serum albumin, which has a molecular mass of 66.5 kDa and a p~ 5, as an example of an acidic protein to demonstrate that paper-based cationic ITP combined with immunobinding can be an effective tool for preconcentrating negatively charged protein. Our results show that the B-Raf IN 1 electrophoretic mobility of BSA is negligible under cationic ITP conditions. However, in the presence of the BSA-specific monoclonal antibody, fluorescently labeled BSA starts migrating toward the anode on a fiberglass paper strip. Adding a secondary antibody conjugated with amine-rich quantum dots (QD) to the sample further increases the electrophoretic mobility of BSA and improves the stacking efficiency of the protein, suggesting B-Raf IN 1 the effectiveness of immunobinding at increasing the electrophoretic mobility of an acidic protein under cationic ITP conditions. The approach developed in this study paves a new avenue to the utilization of cationic ITP to preconcentrate a broad range of target proteins, while simultaneously depleting the abundant proteins in serum samples. Therefore, the technique is potentially useful for developing cationic ITP-based multiplex PADs for POC applications. 2.?Materials and methods 2.1. Chemicals and samples All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Mouse monoclonal to SORL1 Fluor 488 NHS Ester, tetramethylrhodamine-5-isothiocyanate, rabbit anti-mouse IgG labeled with Alexa Fluor 488, and BSA polyclonal antibody were purchased from ThermoFisher. TriLite? fluorescent nanocrystal 540-nm amine (QDs-540-Amine) was purchased from Cytodiagnostics, Inc. (Burlington, Ontario). Mouse monoclonal antibody to human cardiac troponin T (cTnT), rabbit polyclonal B-Raf IN 1 antibody to human cTnT, and mouse monoclonal antibody against BSA was purchased from Abcam (Cambridge, MA). 2.2. Protein labeling BSA was labeled with tetramethylrhodamine-5-isothiocyanate at a ratio of 1 1:2 at pH 8.6. The labeled mixture was passed four times before measurements through a DEAE Sepharose (GE Healthcare) column to remove extra fluorescent probes and dialysis against PBS. Unlabeled goat anti-mouse IgG (Jackson ImmunoResearch) was conjugated to QDs-540-amine using the appropriate LYNX rapid conjugation kit (AbD Serotec). Human cTnT were over expressed in strain BL21(DE3) cells and purified according to our previous studies  and then labeled Alexa Fluor 488, with a ratio of 1 1:2 at pH 8.6, the labeled mixture was first passed a DEAE Sepharose (GE Healthcare) column to remove extra fluorescent probe and dialysis against PBS for four times before measurements. 2.3. LE and TE solutions The LE solution was prepared by adjusting 20 mmol/L KOH.