Western blotting via proximity ligation for high performance protein analysis.

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor ß by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

DNA-assisted protein detection technologies.

Improved protein assays promise to offer new insights into biological processes as well as the identification of new, clinically important biomarkers. In recent years, a number of approaches have been developed where protein-binding reagents, typically antibodies, are equipped with DNA strands to enable protein analyses via powerful nucleic acid detection reactions for improved performance. In this review, we provide a background to this emerging field, and we describe several different ways in which these reagents can improve protein analyses by lowering detection thresholds, improving multiplexing and extending the range of biomolecules available for analysis, both in research settings and in clinical routine.

Sensitive plasma protein analysis by microparticle-based proximity ligation assays.

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

[Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds].

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²âº, Zn²âº, Mn²âº and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

Analysis of genes, transcripts, and proteins via DNA ligation.

Analytical reactions in which short DNA strands are used in combination with DNA ligases have proven useful for measuring, decoding, and locating most classes of macromolecules. Given the need to accumulate large amounts of precise molecular information from biological systems in research and in diagnostics, ligation reactions will continue to offer valuable strategies for advanced analytical reactions. Here, we provide a basis for further development of methods by reviewing the history of analytical ligation reactions, discussing the properties of ligation reactions that render them suitable for engineering novel assays, describing a wide range of successful ligase-based assays, and briefly considering future directions.