Abstract
Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
Highlights
Capillary electrophoresis (CE), coupled with fluorescence detection, liquid chromatography or mass spectrometry, has many applications in separating and detecting biomolecules such as DNA1, metabolites[2], peptides[3] and proteins[4], since it offers high resolution and uses small amounts of samples
The purpose of this study was to investigate if protein detection by capillary isoelectric focusing (IEF) could be enhanced by molecular genetic means involving rolling circle amplification (RCA) (Fig. 1)
Lysates from cultured human umbilical vein endothelial cells (HUVEC) treated with Vascular endothelial growth factor A (VEGFA) were analyzed by capillary isoelectric focusing at dilutions from 25 μg/ml to 3.125 μg/ml
Summary
Capillary electrophoresis (CE), coupled with fluorescence detection, liquid chromatography or mass spectrometry, has many applications in separating and detecting biomolecules such as DNA1, metabolites[2], peptides[3] and proteins[4], since it offers high resolution and uses small amounts of samples. In situ PLA is a variant of the PLA technique, first described by Söderberg et al in 2006, where pairs of antibody-DNA conjugates recognize the same or pairs of interacting proteins of interest and direct the formation of a circular reporter DNA strand[15] This DNA circle is locally amplified via RCA, providing excellent specificity and sensitivity of detection in microscopy[15] and flow cytometry[16]. The state of activity of this signaling pathway can be monitored by studying the phosphorylation of the signal transducing proteins that are present at low concentration in e.g. tissue culture cell lysates or scarce tumor biopsy samples These analyses exemplify the need for assays with minimal sample consumption and excellent specificity for the targeted proteins, provided by the combination of capillary IEF with RCA and in situ PLA
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
