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. 2014 Apr:96:48-53.
doi: 10.1016/j.pep.2014.01.014. Epub 2014 Feb 5.

A semi-automated method for purification of milligram quantities of proteins on the QIAcube

J McGraw  1 , V K Tatipelli  1 , O Feyijinmi  1 , M C Traore  1 , P Eangoor  1 , S Lane  1 , E J Stollar  2
Affiliations

A semi-automated method for purification of milligram quantities of proteins on the QIAcube

J McGraw et al. Protein Expr Purif. 2014 Apr.

Abstract

A growing number of studies require the purification of multiple proteins simultaneously and the development of simple economical high-throughput purification methods is essential. We have tested the purification of two related proteins in a variety of conditions to benchmark the semi-automated affinity chromatography method for the QIAcube that we have developed. We find that this new QIAcube method can successfully purify milligram quantities of proteins with minimal user involvement and performs as well as methods based on gravity. The method could easily be adapted to other chromatography resins and should prove to be a versatile method for optimizing protein expression or purification conditions for multiple proteins while obtaining sufficient amounts for subsequent biochemical analyses.

Keywords: High-throughput automation; Protein expression; Protein purification; QIAcube; SH3 domain.

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Figures

Figure 1
Figure 1
Overview of the QIAcube system. A. Robotic arm with gripper to transfer spin columns and a micro-pipettor to dispense liquids. B. Tips and small volume buffer holder section. C. Micro-centrifuge with 12 buckets to accept rotor adapters (2 are currently shown with spin-column in wash position 1). D. Sample holder/incubator/shaker (2 samples are present). E. Buffer holders (5 are present). The lower panel shows an image and schematic of the rotor adapters.
Figure 2
Figure 2
SDS-PAGE analysis of the flow through (F.T) and first elution (E1) for the purifications outlined in Table 2 (left gel uses gravity column and right gel uses QIAcube, where C and Q refer to Ni-NTA from Clontech and Qiagen respectively). The AbpSH3-ArkA protein band appears slightly higher than AbpSH3 due to its higher molecular weight. The band in the flow through of the Native purifications that is approximately the same position as AbpSh3-ArkA is lysozyme, which is used for cell lysis and does not bind to Ni-NTA.
See this image and copyright information in PMC

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