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Separation and isolation of fusion protein using a new native preparative PAGE device.

Journal of chromatographic science (2012-06-22)
Zhang Xin-Guo Chen Jian-Hua, Yan Lu-Yin, Tang Li, Wang Min, Cheng Dai-Shuang
RESUMEN

A human serum albumin and Thymosin α1 (HSA-Tα1) fusion protein was designed and over-expressed in Pichia pastoris. To purify the fusion protein, a new native preparative electrophoresis system that involved a modified device with a sample receiving chamber, and an assay method with Coomassie Blue G-250 tracing the collection of the protein of interest. In this device, two gels were run in parallel: native vertical collecting polyacrylamide gel electrophoresis (PAGE) and native vertical tracing PAGE. Samples mixed with or without Coomassie Blue G-250 loading buffer were separately loaded to the two aforementioned gels, and the fractions were collected until the tracing protein band combined with dye reached 1 cm from the sample-receiving chamber at the bottom of the gel. Approximately nine fractions were collected at regular intervals of 15 min. HSA-Tα1 fusion protein with 95% relative homogeneity was harvested and manifested similar immunological activities as synthetic Tα1 after a single-step purification of this preparative PAGE. As a result, this system offers a new, rapid and simple method for the purification of the protein of interest.

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Sigma-Aldrich
Brilliant Blue R, 250, for microscopy
Sigma-Aldrich
Brilliant Blue R, pure
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Brilliant Blue R, Dye content ~50 %, Technical grade
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Brilliant Blue R Staining Solution, suitable for (for immunoelectrophoresis protein staining)
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Brilliant Blue R Concentrate, suitable for SDS-PAGE, methanol solution