Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

Cell Chromatography: Biocompatible Chromatographic Separation and Interrogation of Microbial Cells.

The isolation of pure, single colonies lies at the heart of experimental microbiology. However, a microbial colony typically contains around 1 million cells at all stages of the life cycle. Here, we describe a novel cell chromatography method that facilitates the capture, purification, and interrogation of microbial cell populations from both single and mixed cultures. The method described relies on, but is not limited to, differences in surface charge to separate bacterial strains. The method is fully biocompatible, leading to no significant loss of cell viability. The chromatographic capture of cells, combined with selective elution methods, facilitates a greater level of experimental control over the sample inputs required for downstream high-throughput and high-sensitivity analytical methods. The application of the method for interrogating the antibiotic resistance of bacterial strains and for the separation of bacteria from environmental samples is illustrated. IMPORTANCE This is the first report of a method for separating microbial cells using chromatography, with full retention of cell viability. Differences in the surface chemistry of microbial cells provides a means of attracting cells to immobilized microbeads. Some cells are attracted, and some are repelled. The differences in, for example, surface charge can be harnessed to capture, interrogate, and separate environmental samples, thus circumventing the need to use conventional bacterial plating methods. This method will greatly facilitate drug discovery and bioprospecting for novel microbial compounds.

Analytical investigation of the properties and uses of a new hydrophobic molecular sieve.

Distribution coefficients and capacities have been determined for many different organic compounds on a new molecular sieve called silicalite, which does not adsorb water but does adsorb small organic molecules with diameters up to 6 A, from both liquid and gaseous streams. The characteristics of silicalite have been examined closely and new applications for it are briefly described.

Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products.

A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles.

A novel technique for rapid automated genotyping of DNA polymorphisms in the mouse.

The ability to rapidly and reliably genotype mice is an important concern. Traditional methods employ labour intensive and time consuming techniques such as test crossing, gel electrophoresis or nucleic acid hybridization. Here we show that a new molecular biology workstation, the WAVE DNA Fragment Analysis System, can easily resolve polymerase chain reaction (PCR) products that have small differences in their lengths. Analysis is fully automated and takes less than 7 min per sample. Approximately 200 samples can be analysed per day with only minutes of hands-on time after completion of the PCR. Genotyping with the WAVE DNA Fragment Analysis System is a fast and efficient method with minimal manual intervention.

Capillary electrophoresis of peptides and proteins in fused-silica capillaries coated with derivatized polystyrene nanoparticles.

High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 microm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with alpha-omega-diamines such as ethylenediamine or 1,10-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to 1900000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.