Size exclusion chromatography (SEC) is one technique used to separate molecules based on size, and it is one of the main methods used for verification of protein multimer formation. However, this technique can not distinguish proteins from ones with similar molecular sizes. Because smaller molecules can enter pores of materials packed in an SEC column, but larger molecules cannot. Therefore, larger molecules elute earlier than smaller molecules. In the case of target proteins in serum or culture supernatant, the samples have to be purified before SEC analysis. This article describes a seamless process of target proteins for purification, fractionation, and re-injection utilizing a liquid handler.
The liquid handler (LH-40) serves as an autosampler as well as a fraction collector for the LC system. That means samples fractionated during the first run can be injected directly for the second run without transferring them from a fraction collector to an autosampler. For example, with this system (Fig. 1 and 2), the target protein is purified by an affinity column and fractionated as the first step, and then the fractionated protein elution is re- injected for SEC analysis as the second step. These two steps can be performed simply by specifying the method and fraction. In this example, we evaluated IgG in human plasma using an LH-40 liquid handler installed in a Prominence inert LC system.
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