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Zhu Zhu, Keith Roby and Graham Threadgill, Beckman Coulter Inc. A critical issue in many proteomic applications is the availability of large numbers of different purified proteins in sufficient quantity. In order to provide a time- and cost-effective way for high-throughput sample preparation, automated methods to purify and quantitate His-tagged proteins in a 96-well format have been developed. This automated system uses Biomek® 3000 and Qiagen's Ni-NTA Superflow 96 BioRobot* Kit for His-tagged protein purification and the Bio-Rad Protein Assay for protein quantitation. The automated methods allow for the processing of one 96-well plate under either native or denaturing conditions from the bacterial cell pellets. Instrument: Biomek 3000 integrated with the DPC MicroMix 5 Shaker
Hardware: Decks Protein Purification Deck Protein Assay Deck BioMek Software: MethodsThe purification method consists of four steps: lysis of bacterial cell pellets, binding of the His-tagged proteins to the Ni-NTA resin, washing, and elution. The process of purifying one full 96-well plate takes less than 90 minutes. The Bio-Rad protein assay method requires one plate to generate a standard curve and two duplicate sample plates for analysis. The absorbance at 595 nm for the standard and sample plates can be measured from 5 to 60 minutes after the last step on the Biomek 3000. The process to measure the protein concentration from samples in one full 96-well plate takes less than 15 minutes. The variables in the Start step of the methods prompt the user to clarify the number of samples to run (from 8 to 96, in multiples of 8) and whether to run under native or denaturing conditions. Protein Purification Method Protein Assay Method Data: A soluble protein (BirA) and an insoluble protein (PI-6) were purified under native and denaturing conditions, respectively.
Purified proteins were analyzed by Coomassie gel. The yield from a 2 ml bacterial culture was 31 µg for BirA and 43.2 µg for PI-6, as shown above. The band labeled Lysozyme was co-purified as a contaminant from the bacterial lysis step for the BirA. Legend: M = marker, 1 = BirA, 2 = PI-6. * All trademarks are the property of their respective owners. For Research Use Only; not for use in diagnostic procedures. For comments or questions about T3 Update, please contact David Daniels, Ph.D., editor.
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