Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE

Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles

Measurement and prediction of bark thickness in Picea abies: Assessment of accuracy, precision, and sample size requirements

Tree and log diameters are usually measured outside bark, but inside-bark diameters are of greater economic interest and are often derived with local or regional bark thickness equations. To date, the influence of measurement method, sampling design, and sample size on bark thickness equation accuracy and precision has received limited attention. The objectives of this study were to use an extensive regional bark thickness dataset for Norway spruce (Picea abies [L.] Karst) in south-western Germany to: (1) quantify accuracy and precision of bark thickness measurements with a Swedish bark gauge; (2) determine the required number of measurements to assess the within-tree variation, and (3) estimate required sample sizes per plot and region to develop an accurate bark thickness prediction equation. Bark gauge readings were validated with measurements derived from X-ray computed tomography (CT) and indicate that Swedish bark gauges generally overestimated bark thickness by 13.6 ± 28.4% (mean ± SD). Results suggested having at least one measurement location every two meters along a tree bole and at least five bark thickness measurements per each of these locations to achieve an allowable error ofThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Potentially increased sawmill yield from hardwoods using X-ray computed tomography for knot detection

International audienceAbstractContextOne of the most important wood defects affecting the value yield from European beech (Fagus sylvatica [L.]) logs is knots that are visible on the sawn wood surface. The non-invasive technology of X-ray computed tomography (CT) can be used for the assessment of log internal features, especially the geometry and position of knots before primary breakdown to support the decision of value-optimised log rotation in sawmills.AimsThe objective of this study was to test whether value-optimised log rotation can be performed successfully by using the CT-derived knowledge of internal knottiness for the hardwood species beech.MethodsSize parameters of 670 knots were measured and their position was marked in CT images from 33 logs. The 3D-reconstructed logs were virtually sawn in 12 different rotational angles using the software InnoSIM. This allowed visual grading of the simulated sawn wood and the calculation of product volume and value.ResultsThe results show that if optimal rotation was applied to each single log, both total volume as well as total product value yield could be improved by up to 24 % compared with the average yield of all simulated rotational angles.ConclusionIn this small-scale study, it is demonstrated that CT technology could be used to support the decision about optimal rotational angle of beech logs to maximise volume and value yield

Conformation space of a heterodimeric ABC exporter under turnover conditions

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters