Summary of the April 2013 issue of BioTechniques
The April 2013 issue of BioTechniques will feature articles describing new methods for: (i) enhanced, speedy isolation of endogenous protein complexes, (ii) production of single stranded DNA products using deoxyribozymes and rolling circle amplification, (iii) generation of stable cell lines for protein-protein interaction analysis, (iv) determination of the number and arrangement of cell surface proteins, and (v) isolation of target proteins using a novel calcium-responsive tag. In addition, the April issue will also feature a special Tech News article that takes a peek inside the current toolbox of the genetic engineer, examining how ZFNs, TALENs, homologous recombination and other techniques can be applied to different genome engineering projects.
Isolating endogenous protein complexes is important for understanding how proteins interact and function within cells. When isolating such complexes, it is often critical to use gentle conditions and perform experiments as rapidly as possible. Two articles in this issue of BioTechniques report on new tools to assist in the isolation of tagged proteins. First, a team of researchers detail their modification to an existing peptide that enables the rapid elution of protein-A tagged molecules in less than 15 minutes. In comparison to the previous protein-A peptide, the author’s newly modified reagent is significantly faster (15 minutes compared to several hours for elution), works under milder conditions, and can be removed following elution using a simple spin column procedure. This new peptide should enhance workflows for researchers purifying proteins, and proteins complexes, with protein-A tags.
The second April article focused on protein tagging and isolation describes a new calcium responsive tag. Here, the authors have developed a calcium-stimulus responsive synthetic peptide tag based on the naturally occurring repeat-in-toxin (RTX) domain, which can be used for protein precipitation and purification under gentle, calcium-induced conditions. This novel tag provides researchers a new, milder alternative when looking for a stimulus responsive tag for their protein isolations.
Protein interaction methods such as BRET and FRET rely on transient transfection of potential protein interaction partners. Transient transfections can result in over-expression of interacting partners, as well as non-homogeneous expression, complicating the interpretation of protein interaction assays. But in April, a team of researchers present a new method for the rapid generation of stable cell lines that provide single copy, isogenic expression of proteins for BRET interaction analyses.
Determining the stoichiometry of membrane proteins has traditionally been done through stochastic GFP photobleaching using total internal reflection (TIRF) microscopy. However, TIRF microscopy requires specialized equipment and knowledge, limiting usage. In the April 2013 issue of BioTechniques, a group of researchers describe the application of laser scanning confocal microscopy with a membrane sheet preparation to assess membrane protein oligomerization and distribution through GFP photobleaching. The simplicity of the author’s technique should enable GFP photobleaching experiments to be performed on membrane proteins using many different epifluorescence microscopy systems, thus significantly extending the potential use of this technique.
Generating single stranded DNA for molecular biology applications can be a challenge for scientists, but in the April issue, a team from Yale University describes a new approach to obtaining ssDNA of specific lengths. By incorporating deoxyribozyme sequences into templates for rolling circle amplification, the authors clearly show that single stranded DNAs of single and multiple-unit lengths with defined sizes and precise termini can be obtained. The authors go on to demonstrate the utility of their deoxyribozyme incorporated sequences by using the technique to generate a set of ssDNA markers.
Finally, the April issue will also feature a special Tech News examining the growing toolkit for engineering the genome. Here, the latest techniques (including ZFNs, TALENs, CRISPRs, MAGE, and homologous recombination) will be highlighted and compared with the intent of providing readers with a clearer understanding of the possibilities and potential of each techniques through specific examples. In addition to the Tech News, our other regular features such as the Scientist Profile, BioSpotlights, Troubleshooting Forum and Citations will also be included in the April issue.
Keywords: genome engineering, protein isolation and purification, chromatography, calcium-responsive tags, protein tags, ZFN, TALEN, MAGE, TIRF, confocal microscopy, rolling circle amplification (RCA), ribozymes, single-stranded DNA markers, GFP photobleaching, single molecule analysis, BRET, FRET, protein-A, generation of stable cell lines, transfection