An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 microM of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10,000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of the DNA analyte concentration. Using a combination of spectrofluorometry and flow cytometry experiments, we found that PicoGreen exhibited better overall performance than all the tested dsDNA binding dyes, such as TOTO-1. Fluorometric titration revealed that typical DNA staining protocols designed on the basis of the dye/bp ratio were highly dependent upon the DNA concentration for optimal results. PicoGreen was the least sensitive to the solution dye/bp ratio and was highly fluorescent in the presence of dsDNA. Using this new protocol, accurate histograms of HindIII digested lambda DNA were demonstrated for DNA concentrations ranging from 5 to 2000 ng/mL, and for dye/bp ratios from 106:1 to 1:4 at 0.8 microM of PicoGreen. The new one-step protocol is broadly applicable to any sensitive, laser-induced fluorescence method for detection of nucleic acids.
Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...
It is generally accepted that plasmids containing the same origin of replication are incompatible. We have re-examined this concept in terms of the plasmid copy number, by introducing plasmids containing the same origin of replication and different antibiotic resistance genes into bacteria. By selecting for resistance to only one antibiotic, we were able to examine the persistence of plasmids carrying resistances to other antibiotics. We find that plasmids are not rapidly lost, but are able to persist in bacteria for multiple overnight growth cycles, with some dependence upon the nature of the antibiotic selected for. By carrying out the experiments with different origins of replication, we have been able to show that higher copy number leads to longer persistence, but even with low copy plasmids, persistence occurs to a significant degree. This observation holds significance for the field of protein engineering, as the presence of two or more plasmids within bacteria weakens, and confuses, the connection between screened phenotype and genotype, with the potential to wrongly assign specific phenotypes to incorrect genotypes.
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