Automated Polyhistidine-Tagged Protein Purification Using the ...

Automated His-Tagged Protein Purification ... continued250 –150 –100 –75 –50 –35 –25 –15 –––10 –M1 2 3 4 5 6 7 8F E F E F E F E M F E F E F E F EFigure 5. Automated protein purification of polyhistidine-taggedthermostable firefly luciferase on the Kingfisher ® using the MagneHisProtein Purification System. Eight samples of bacteria expressing polyhistidinetaggedthermostable firefly luciferase were cultured. For each sample, purified proteinwas eluted from the MagneHis Ni-Particles (lanes E), and the flowthrough fractions ofproteins not captured by the MagneHis Ni-Particles were collected (lanes F). Lane Mcontains the Broad Range Protein Molecular Weight Markers (Cat.# V8491).Instrument Configuration and ThroughputConsiderationsThe Biomek ® FX workstation processed all 96 samplessimultaneously due to the 96-channel PODconfiguration. This increased sample throughputconsiderably. Our Tecan Genesis ® RSP 150 model wasequipped with 8 standard fixed tips and, like theBiomek ® 2000, can pipet 8 samples at a time, therebyincreasing the flexibility in the number of samplesprocessed during a single run but decreasing overallsample throughput (Table 1). A Tecan Genesis ® RSP witha TeMo 96-channel pipetting tool would result inthroughput similar to that of the Biomek ® FXworkstation.Robotic throughput varies depending on the instrumentand its configuration. While it is possible to processsamples faster manually than with the Biomek ® 2000 orTecan Genesis ® RSP workstations (Table 1), manualprocessing of 96 samples at a time is labor intensive,tedious and prone to error.3927TA12_2ATable 1. MagneHis Protein Purification System Throughput.Time per Time per Throughput inMethod 96-Well Plate Sample 8-Hour ShiftBiomek ® FX 25 minutes 15 seconds 20 platesBiomek ® 2000 90 minutes 60 seconds 5 platesGenesis ® RSP 90 minutes 60 seconds 5 platesKingfisher ® NA 1 50 seconds 6 plates 2Manual 45 minutes 3 30 seconds 8 plates1Two Kingfisher ® plates, each containing 12 samples, processed simultaneously in 20 minutesduring a single automated run.2Forty-eight plates, each containing 12 samples (576 samples total) in an 8-hour shift. Equivalentto six 96-well plates.3Approximate.The Thermo Labsystems Kingfisher ® is a very practicalinstrument for processing small numbers of samples inan automated format relatively quickly without userintervention (Table 1). Two plates of 12 samples each canbe processed simultaneously with this instrument. Thefollowing reagents must be dispensed manually intoeach plate prior to the run:Row A: MagneHis Ni-ParticlesRow B: Cell pellets resuspended in MagneHis CellLysis ReagentRow C: MagneHis Binding/Wash BufferRow D: MagneHis Binding/Wash BufferRow E: MagneHis Binding/Wash BufferRow F: MagneHis Elution BufferConclusionsWe have automated the MagneHis Protein PurificationSystem for the rapid purification of polyhistidine-taggedproteins on several automated workstations. Theinnovative MagneHis Cell Lysis Reagent eliminates theneed for manual intervention (no sonication orcentrifugation steps during the purification process) andallows for a truly walkaway protein purification system.Methods have been developed for the Biomek ® 2000,Biomek ® FX, Tecan Genesis ® RSP and Kingfisher ®automated platforms. These methods allow a quick startupin your laboratory and offer several key advantagesfor end-users. These advantages include a simple processthat is easily automated on a variety of platforms forhigh-throughput applications, chemistry that is easilyscaled up for larger sample volumes and high-qualitypurified proteins with few background bands.Reference1. Godat, B. et al. (2003) Promega Notes 83, 2–58

Protocols◆ MagneHis Protein Purification System Technical Manual #TM060,Promega Corporation(www.promega.com/tbs/tm060/tm060.html)Michael Bjerke, M.S.AutomatedSolutions ScientistLaurie Orr, B.S.Research ScientistNotPicturedCristopher Cowan,Ph.D.AutomatedSolutions SpecialistOrdering InformationProduct Size Cat.#MagneHis ProteinPurification System (a,b) 2ml V850010ml V8550MagneHis Ni-Particles (a,b) 2ml V856010ml V8565MagnaBot ® 96 MagneticSeparation Device 1 each V8151MagnaBot ® Adapter T1 1 each V8481(a)Patent Pending.(b)Certain applications of this product are covered by patents issued and applicable in certaincountries. Because purchase of this product does not include a license to perform any patentedapplication, users of this product may be required to obtain a patent license depending upon theparticular application and country in which the product is used.(c)U.S. Pat. No. 5,552,302, Australian Pat. No. 646803 and other patents.(d)U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, Australian Pat. Nos. 616881 and 641261 andother pending and issued patents, which claim vectors encoding a portion of human placentalribonuclease inhibitor, are exclusively licensed to Promega Corporation.MagnaBot and RNasin are trademarks of Promega Corporation and areregistered with the U.S. Patent and Trademark Office. MagneHis is atrademark of Promega Corporation.Biomek is a registered trademark of Beckman Instruments, Inc. BioWorks isa trademark of Beckman Coulter, Inc. Genesis is a registered trademark ofTecan AG Corporation. Kingfisher is a registered trademark of LabsystemsOy. Micromix is a registered trademark of Clintec Nutrition Co.Becky Godat, M.A.Research Scientist9Promega Notes www.promega.com Number 83 2003