Purification by electrophoresis

Step 5 The final step is removal of all protecting groups and cleavage of the ester bond holding the DNA to the silica. All these reactions are done at the same time by treatment with aqueous NH3. Purification by electrophoresis then yields the synthetic DNA. 

We typically use RNA purification by the Trizol method (Invitrogen, following the manufacturer s instructions), which has the advantage over column-based methods that it can purify small amounts of RNA and retain miRs. Purified RNA is dissolved in RJNAse-free water and stored at —80°. RNA quality is assessed on an Agilent Bioanalyzer (Agilent Technologies) or by gel electrophoresis. 

The purification step by 2-D electrophoresis implies the use of denaturing conditions (SDS), and thus, is not appropriate for obtaining antibodies directed against native structures. For that purpose, the protein should be transferred onto nitrocellulose after purification by classical nondenaturing methods and gel electrophoresis under nondenaturing conditions. 

The acid-base properties, and hence ionic character, of peptides and proteins also can be used to achieve separations. Ion-exchange chromatography, similar to that described for amino acids (Section 25-4C), is an important separation method. Another method based on acid-base character and molecular size depends on differential rates of migration of the ionized forms of a protein in an electric field (electrophoresis). Proteins, like amino acids, have isoelectric points, which are the pH values at which the molecules have no net charge. At all other pH values there will be some degree of net ionic charge. Because different proteins have different ionic properties, they frequently can be separated by electrophoresis in buffered solutions. Another method, which is used for the separation and purification of enzymes, is affinity chromatography, which was described briefly in Section 9-2B. 

Figure 2 SDS-PAGE analysis of purified HRV14 2A and 3C proteases. Protein samples (—1-2 (ig) were separated by electrophoresis on 16% gels and then stained with Coomas-sie blue. Lanes 1-4 represent the purification ofHRV14 2A protein. Lanes 1, transformed cell lysate 2, urea-solubilized inclusion bodies 3 and 4, 2A protease preparations after Mono Q and Superdex-75 columns, respectively. Lane 5 represents the purified HRV14 3C protease.

In some instances, direct paper chromatography or paper electrophoresis of the crude extracts has been used, but preliminary purification by heavy-metal precipitation is then often desirable. 

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