Source: Laboratory of Dr. Ana J. García-Sáez — University of Tübingen
Most samples of interest are mixtures of many different components. Sample preparation, a key step in the analytical process, removes interferences that may affect the analysis. As such, developing separation techniques is an important endeavor not just in academia, but also in industry.
One way to separate mixtures is to use their solubility properties. In this short paper, we will deal with aqueous solutions. The solubility of a compound of interest depends on (1) ionic strength of solution, (2) pH, and (3) temperature. By manipulating with these three factors, a condition in which the compound is insoluble can be used to remove the compound of interest from the rest of the sample.1…
Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a…
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ion-exchange chromatography is a type of chromatography that separates analytes based on charge. A column is used that is filled with a charged stationary phase on a solid support, called an ion-exchange resin. Strong cation-exchange chromatography preferentially separates out cations by using a negatively-charged resin while strong anion-exchange chromatography preferentially selects out anions by using a positively-charged resin. This type of chromatography is popular for sample preparation, for example in the cleanup of proteins or nucleic acid samples.
Ion-exchange chromatography is a two-step process. In the first step, the sample is loaded onto the column in a loading buffer. The binding of the charged sample to the column resin is based on ionic interactions of the resin to attract the sample of the opposite charge. Thus, charged samples of opposite polarity to the resin are strongly bound. Other molecules that are not charged or are of the opposite charge are not bound and are washed through the column. The second step is to elute the analyte that is bound to the resin. This is accomplished with a salt gradient, where the amount of salt in the buffer is slowly increased. Fractions are collected at the end of the column as…