Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins

Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro

Dependence of Maximum Trappable Field on Superconducting Nb3Sn Cylinder Wall Thickness

Uniform dipole magnetic fields from 1.9 to 22.4 kOe were permanently trapped, with high fidelity to the original field, transversely to the axes of hollow Nb3Sn superconducting cylinders. These cylinders were constructed by helically wrapping multiple layers of superconducting ribbon around a mandrel. This is the highest field yet trapped, the first time trapping has been reported in such helically wound taped cylinders, and the first time the maximum trappable field has been experimentally determined as a function of cylinder wall thickness.Comment: 8 pages, 4 figures, 1 table. PACS numbers: 74.60.Ge, 74.70.Ps, 41.10.Fs, 85.25.+

Atomic Hydrogen Cleaning of Polarized GaAs Photocathodes

Atomic hydrogen cleaning followed by heat cleaning at 450$^\circ$C was used to prepare negative-electron-affinity GaAs photocathodes. When hydrogen ions were eliminated, quantum efficiencies of 15% were obtained for bulk GaAs cathodes, higher than the results obtained using conventional 600$^\circ$C heat cleaning. The low-temperature cleaning technique was successfully applied to thin, strained GaAs cathodes used for producing highly polarized electrons. No depolarization was observed even when the optimum cleaning time of about 30 seconds was extended by a factor of 100

Surface Analysis of OFE-Copper X-Band Accelerating Structures and Possible Correlation to RF Breakdown Events

X-band accelerator structures meeting the Next Linear Collider (NLC) design requirements have been found to suffer vacuum surface damage caused by radio frequency (RF) breakdown, when processed to high electric-field gradients. Improved understanding of these breakdown events is desirable for the development of structure designs, fabrication procedures, and processing techniques that minimize structure damage. RF reflected wave analysis and acoustic sensor pickup have provided breakdowns localization in RF structures. Particle contaminations found following clean autopsy of four RF-processed travelling wave structures, have been catalogued and analyzed. Their influence on RF breakdown, as well as that of several other material-based properties, will be discussed.Comment: 21 pages, 8 figures, 4 tables, Submitted to JVST A as a proceeding of the 50th AVS conference (Baltimore, MD, 2-7 Nov 2003

Beam Test of a Segmented Foil SEM Grid

A prototype Secondary-electron Emission Monitor (SEM) was installed in the 8 GeV proton transport line for the MiniBooNE experiment at Fermilab. The SEM is a segmented grid made with 5 um Ti foils, intended for use in the 120 GeV NuMI beam at Fermilab. Similar to previous workers, we found that the full collection of the secondary electron signal requires a bias voltage to draw the ejected electrons cleanly off the foils, and this effect is more pronounced at larger beam intensity. The beam centroid and width resolutions of the SEM were measured at beam widths of 3, 7, and 8 mm, and compared to calculations. Extrapolating the data from this beam test, we expect a centroid and width resolutions of 20um and 25 um, respectively, in the NuMI beam which has 1 mm spot size.Comment: submitted to Nucl. Instr. Meth.

Pion-Muon Asymmetry Revisited

Long ago an unexpected and unexplainable phenomena was observed. The distribution of muons from positive pion decay at rest was anisotropic with an excess in the backward direction relative to the direction of the proton beam from which the pions were created. Although this effect was observed by several different groups with pions produced by different means, the result was not accepted by the physics community, because it is in direct conflict with a large set of other experiments indicating that the pion is a pseudoscalar particle. It is possible to satisfy both sets of experiments if helicity-zero vector particles exist and the pion is such a particle. Helicity-zero vector particles have direction but no net spin. For the neutral pion to be a vector particle requires an additional modification to conventional theory as discussed herein. An experiment is proposed which can prove that the asymmetry in the distribution of muons from pion decay is a genuine physical effect because the asymmetry can be modified in a controllable manner. A positive result will also prove that the pion is NOT a pseudoscalar particle.Comment: 9 pages, 3 figure

Nuclear Magnetic Quadrupole Moments in Single Particle Approximation

A static magnetic quadrupole moment of a nucleus, induced by T- and P-odd nucleon-nucleon interaction, is investigated in the single-particle approximation. Models are considered allowing for analytical solution. The problem is also treated numerically in a Woods-Saxon potential with spin-orbit interaction. The stability of results is discussed.Comment: LATEX, 9 pages, 1 postscript figure available upon request from "[email protected]". BINP 94-4

Orbital Debris-Debris Collision Avoidance

We focus on preventing collisions between debris and debris, for which there is no current, effective mitigation strategy. We investigate the feasibility of using a medium-powered (5 kW) ground-based laser combined with a ground-based telescope to prevent collisions between debris objects in low-Earth orbit (LEO). The scheme utilizes photon pressure alone as a means to perturb the orbit of a debris object. Applied over multiple engagements, this alters the debris orbit sufficiently to reduce the risk of an upcoming conjunction. We employ standard assumptions for atmospheric conditions and the resulting beam propagation. Using case studies designed to represent the properties (e.g. area and mass) of the current debris population, we show that one could significantly reduce the risk of nearly half of all catastrophic collisions involving debris using only one such laser/telescope facility. We speculate on whether this could mitigate the debris fragmentation rate such that it falls below the natural debris re-entry rate due to atmospheric drag, and thus whether continuous long-term operation could entirely mitigate the Kessler syndrome in LEO, without need for relatively expensive active debris removal.Comment: 13 pages, 8 figures. Accepted for publication in Advances in Space Researc