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Schulz Ulriksen posted an update 6 months ago
Adherent cells are easily prepared for cell staining by growing on a suitable microscope slide, coverslip, or plastic tissue culture dish. For high-resolution studies, adherent cells should be grown on the highest available grade glass coverslips, because the controlled thickness, flatness, and good optical properties of a proper coverslip are required to produce the best images. In addition, the glass surface is compatible with all fixing and staining solutions. If many antibodies, different dilutions, or various controls are to be tested on the same cell type, plating the cells onto multiwell slides can be helpful. For low-resolution work, such as crude antigen detection, hybridoma screening, or antibody titration, cells for staining can be grown on regular tissue culture dishes.Chromatin immunoprecipitation, commonly referred to as ChIP, is a powerful technique for the evaluation of in vivo interactions of proteins with specific regions of genomic DNA. Formaldehyde is used in this technique to cross-link proteins to DNA in vivo, followed by the extraction of chromatin from cross-linked cells and tissues. Harvested chromatin is sheared and subsequently used in an immunoprecipitation incorporating antibodies specific to protein(s) of interest and thus coprecipitating and enriching the cross-linked, protein-associated DNA. The cross-linking process can be reversed, and protein-bound DNA fragments of optimal length ranging from 200 to 1000 base pairs (bp) can subsequently be purified and measured or sequenced by numerous analytical methods. In this protocol, two different fixation methods are described in detail. The first involves the standard fixation of cells and tissue by formaldehyde if the target antigen is highly abundant. The dual cross-linking procedure presented at the end includes an additional preformaldehyde cross-linking step and can be especially useful when the target protein is in low abundance or if it is indirectly associated with chromatin DNA through another protein.AAV virions are built from three major capsid proteins, VP1, VP2, and VP3, at a ratio of 1118. On a silver-stained SDS-polyacrylamide gel, VP1, VP2, and VP3 should be the only visible bands in a highly purified recombinant adeno-associated virus (rAAV) preparation, migrating at approximately 87, 73, and 62 kDa, respectively. see more This protocol describes how SDS-PAGE and silver staining can be used to determine the purity of an rAAV preparation. In addition, using a highly purified rAAV preparation whose particle titer is known, this assay can be used to derive a semiquantitative estimate of the particle concentration of a test vector.Negative staining is a simple and rapid method for studying the morphology and ultrastructure of small particulate specimens (e.g., viruses, bacteria, cell fragments, and isolated macromolecules such as proteins and nucleic acids). The technique described in this protocol involves allowing particles or fragments of cells to settle onto a support film, then applying a drop of metal salt solution to the adherent particulate specimen. The stain penetrates the interstices of the particles to bring out detail. In this situation, the preparation dries rapidly. The dissolved substance precipitates out of solution in an amorphous condition at the 0.1-nm level, and it is deposited over the support film and exposed surface of the specimen. The theoretical requirements of a good negative staining are a substance (1) of high density to provide high contrast, (2) at high solubility so that the stain does not come out of solution prematurely but does so only at the final stage of drying, (3) of high melting point and boiling point so that the material does not evaporate at high temperatures induced by the electron beam, and (4) in which the precipitate should be essentially amorphous down to the limit of resolution.Centrifugation to equilibrium in cesium chloride gradients has been used for more than 40 yr to purify viruses. The application of high G-forces for a long period of time to a solution of CsCl generates a density gradient that allows separation of empty, partially packaged, and fully packaged viral particles from cellular debris, proteins, and nucleic acids in the crude viral lysate on the basis of their buoyant densities. This protocol describes the use of CsCl gradients to purify AAV vectors from crude viral lysates.Photosystem II (PS II) captures solar energy and directs charge separation (CS) across the thylakoid membrane during photosynthesis. The highly oxidizing, charge-separated state generated within its reaction center (RC) drives water oxidation. Spectroscopic studies on PS II RCs are difficult to interpret due to large spectral congestion, necessitating modeling to elucidate key spectral features. Herein, we present results from time-dependent density functional theory (TDDFT) calculations on the largest PS II RC model reported to date. This model explicitly includes six RC chromophores and both the chlorin phytol chains and the amino acid residues less then 6 Å from the pigments’ porphyrin ring centers. Comparing our wild-type model results with calculations on mutant D1-His-198-Ala and D2-His-197-Ala RCs, our simulated absorption-difference spectra reproduce experimentally observed shifts in known chlorophyll absorption bands, demonstrating the predictive capabilities of this model. We find that inclusion of both nearby residues and phytol chains is necessary to reproduce this behavior. Our calculations provide a unique opportunity to observe the molecular orbitals that contribute to the excited states that are precursors to CS. Strikingly, we observe two high oscillator strength, low-lying states, in which molecular orbitals are delocalized over ChlD1 and PheD1 as well as one weaker oscillator strength state with molecular orbitals delocalized over the P chlorophylls. Both these configurations are a match for previously identified exciton-charge transfer states (ChlD1+PheD1-)* and (PD2+PD1-)*. Our results demonstrate the power of TDDFT as a tool, for studies of natural photosynthesis, or indeed future studies of artificial photosynthetic complexes.
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