22 research outputs found
Selection of aptamers for a protein target in cell lysate and their application to protein purification
Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies
Single-molecule techniques in biophysics : a review of the progress in methods and applications
Single-molecule biophysics has transformed our understanding of the
fundamental molecular processes involved in living biological systems, but also
of the fascinating physics of life. Far more exotic than a collection of
exemplars of soft matter behaviour, active biological matter lives far from
thermal equilibrium, and typically covers multiple length scales from the
nanometre level of single molecules up several orders of magnitude to longer
length scales in emergent structures of cells, tissues and organisms.
Biological molecules are often characterized by an underlying instability, in
that multiple metastable free energy states exist which are separated by energy
levels of typically just a few multiples of the thermal energy scale of kBT,
where kB is the Boltzmann constant and T the absolute temperature, implying
complex, dynamic inter-conversion kinetics across this bumpy free energy
landscape in the relatively hot, wet environment of real, living biological
matter. The key utility of single-molecule biophysics lies in its ability to
probe the underlying heterogeneity of free energy states across a population of
molecules, which in general is too challenging for conventional ensemble level
approaches which measure mean average properties. Parallel developments in both
experimental and theoretical techniques have been key to the latest insights
and are enabling the development of highly-multiplexed, correlative techniques
to tackle previously intractable biological problems. Experimentally,
technological developments in the sensitivity and speed of biomolecular
detectors, the stability and efficiency of light sources, probes and
microfluidics, have enabled and driven the study of heterogeneous behaviours
both in vitro and in vivo that were previously undetectable by ensemble
methods..
'Inject-Mix-React-Separate-and-Quantitate' (IMReSQ) method for screening enzyme inhibitors
Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors
Ultra-scaled Z-RAM cell
Ultra-scaled Z-RAM cells based on MuGFETs are demonstrated for the first time. Effects of physical parameters such as channel doping concentration, fin width, and gate length on Z-RAM cell performance are discussed. Transient measurements and simulations prove that the basic operational principles are effective on Z-RAM cells with a gate length down to 12.5 nm