Impact of Immunization Technology and Assay Application on Antibody Performance – A Systematic Comparative Evaluation

Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost (“DNA immunization”; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used

Plasma and cellular fibronectin: distinct and independent functions during tissue repair

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes

Atomic-resolution imaging of surface and core melting in individual size-selected Au nanoclusters on carbon

Experimental and theoretical data sets for the Nature Communications paper "Atomic-resolution imaging of surface and core melting in individual size-selected Au nanoclusters on carbon". Data structure and formats are described in the README.docx file

Progress in understanding disruptions triggered by massive gas injection via 3D non-linear MHD modelling with JOREK

3D non-linear MHD simulations of a D 2 massive gas injection (MGI) triggered disruption in JET with the JOREK code provide results which are qualitatively consistent with experimental observations and shed light on the physics at play. In particular, it is observed that the gas destabilizes a large m/n = 2/1 tearing mode, with the island O-point coinciding with the gas deposition region, by enhancing the plasma resistivity via cooling. When the 2/1 island gets so large that its inner side reaches the q = 3/2 surface, a 3/2 tearing mode grows. Simulations suggest that this is due to a steepening of the current profile right inside q = 3/2. Magnetic field stochastization over a large fraction of the minor radius as well as the growth of higher n modes ensue rapidly, leading to the thermal quench (TQ). The role of the 1/1 internal kink mode is discussed. An I p spike at the TQ is obtained in the simulations but with a smaller amplitude than in the experiment. Possible reasons are discussed