Summary of the January 2013 issue of BioTechniques
The January 2013 issue of BioTechniques will feature peer-reviewed research articles describing methods for determining both membrane protein stoichiometry and insertion patterns, a new phase changing peptide for bioseparations, a novel PCR approach for enhanced sensitivity, and an novel analysis tool to deal with missing data associated with ‘omics studies. In addition to these research articles, the January issue will also contain a Tech News feature on recent developments and advances in protein separation technologies.
Analysis of membrane proteins is critical as these biomolecules enable a cell to communicate with the surrounding environment. In this issue of BioTechniques, two groups of researchers describe new techniques aimed at understanding the distribution and localization of membrane proteins. In the first article, researchers from Helsinki detail a biochemical technique that takes advantage of a cold-adapted trypsin enzyme and a fluorescent dye to label pools of proteins prior to incorporation in the membrane. In this way, researchers can study trafficking of these proteins to the membrane in response to signals or other cellular cues. The second article is from a group of scientists from Chile describing their development of a cell-free assay capable of determining the stoichiometry of plasma membrane proteins. Here, a methodological modification is introduced allowing stochastic GFP photobleaching to be used with any epifluorescence microscope rather than exclusively needing a total internal fluorescence microscopy, making the technique more accessible and affordable.
Chromatography can be an important step in the purification of proteins, including those found at the membrane. One approach being advanced to improve protein purification is the creation of stimulus-responsive protein tags that activate under specific conditions to enable precipitation of a target protein. In a Report in the January issue, a group of researchers describe a new calcium-responsive precipitation tag that can be easily cleaved following protein isolation. This new tag enables bioseparations under more gentle conditions than previously possible using other approaches.
‘Omics datasets, such as those generated by mass spectrometry, lead to large complex datasets. The problem is that these datasets also include a large number of non-random missing values, making data analysis tools such as principal component analysis (PCA) less effective in pattern visualization. To overcome this limitation and extend analysis of new large-scale datasets, researchers at the Pacific Northwest National Laboratory describe the use of sequential projection pursuit PCA as an alternative to PCA that works even in the presence of missing data without imputation.
Viral diagnostics often requires the quantitative amplification of viral particles from very low starting concentrations, a challenge for traditional PCR techniques. In a Report slated for the January issue, researchers from Oxfordshire, UK describe the development of a PCR technique called Polymerase Chain Displacement Reaction (PCDR) which takes advantage of multiple nested primers to first extend a new strand initiating from an outer primer which in turn then displaces the inner primer. The result of using the multiple combinations of primers suggested in PCDR is the 10-fold increase in sensitivity, enhancing the potential of PCR in viral diagnostics, as well as other applications focused on low copy number materials, in the future.
Keywords: chromatography, sequencing, mass spectrometry, network analysis, PCR, diagnostics, GFP, protein purification and separation, fluorescence microscopy, membrane proteins, membrane trafficking, data analysis, ‘omics approaches and technologies, protein tag