The effects of mobility corrections on carrier ampholytes are studied at various ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Hückel correction to the carrier ampholytes. Two model proteins with ten charge states but without ionic stren...

Because of the outstanding separating capabilities of two-dimensional electrophoresis for complete proteins, it would be advantageous to be able to apply it to all types of proteins. Unfortunately, severe solubility problems hamper the analysis of many classes of proteins, but especially membrane proteins. These problems arise mainly in the extraction and isoelectric focusing steps, and solutio...

High-speed analyses of proteins by microchip isoelectric focusing (MCIEF) were investigated on straight channel chips. By employing a commercially available microchip electrophoresis instrument equipped with a linear-imaging UV detector, sample proteins focused in a separation channel could be detected without a mobilization step. Typically, standard proteins were focused within 210 s at differ...

Efficient fractionation of peptides is an essential prerequisite for comprehensive analysis of complex protein mixtures by shotgun mass spectrometry. The separation of peptides by isoelectric focusing is particularly attractive due to its orthogonality to reverse-phase HPLC. Here, we present a protocol for in-gel peptide isoelectric focusing using immobilized pH gradient strips. The method show...

Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, we have investigated a number of pre-fractionation methods, at both the protein and peptide level, for the characterization of the skelet...

This work presents several critical details for making cIEF-MALDI-MS a robust technique which will allow for more routine application and aid in automation. This includes emphasis on the hardware necessary for syringe pump mobilization and proper protocol for preventing disruption from gas bubbles. Following these guidelines, excellent elution time reproducibility is demonstrated for six pI mar...

We study how gravitational focusing (GF) of dark matter by the Sun affects the annual and biannual modulation of the expected signal in non-directional direct dark matter searches, in the presence of dark matter substructure in the local dark halo. We consider the Sagittarius stream and a possible dark disk, and show that GF suppresses some, but not all, of the distinguishing features that woul...

In addition to the Neurospora crassa mutant col-2, mutations in two other unlinked genes, balloon and frost, lead to distinct morphological growth and abnormal glucose-6-phosphate dehydrogenases. The glucose-6-phosphate dehydrogenases from these strains have increased thermolabilities, altered kinetic parameters, and distinctive electrofocusing patterns. The altered enzyme segregates with each ...

Agarose isoelectric focusing was used to demonstrate oligoclonal bands in cerebrospinal fluid (CSF) and serum from 998 consecutive neurological patients. Compared with agarose electrophoresis, agarose isoelectric focusing was slightly more sensitive, showing more (and more easily discernible) oligoclonal bands. Agarose isoelectric focusing, which has good reproducibility, revealed oligoclonal b...

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ri...