Non-robotic high-throughput setup yielding diffraction-quality crystals in nanoliter drops manually assembled by motorized pipette

R. SKRABANA^1,2, O. CEHLAR¹, M. NOVAK^1,2

¹Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 845 10 Bratislava, Slovakia

²Axon Neuroscience GmbH, Rennweg 95/b, A-1030 Vienna, Austria

Nanoliter-sized drops of protein solution and precipitant are characteristic of high-throughput protein crystallizations screening. Present-day crystallization practice requires almost inevitably the robotized setup for reliable nanoliter crystallization drop pipetting. In this work, a protocol for reproducible manual assembly of protein crystallization screening experiments adopting nanoliter-sized drops in 96-well format has been designed and developed. The protocol exploits the repetitive-pipetting mode of commonly available motorized single and multichannel pipette, together with simple tools available in ordinary laboratory. For assembly of one crystallization screen hawing 96 crystallization conditions with 10 mg/ml of protein in equilibrated drop, less than 350 µg of protein was used. The protocol approach was verified on the finding crystallization conditions for antibody Fab fragments and their complexes with tau protein antigens. Diffraction-quality crystals were grown directly from the screen conditions on 96-well plates and yielded complete diffraction data sets on synchrotron source. The results proved that successful crystallization in nanoliter high-throughput format is affordable even for a small-sized crystallography laboratory, without the need of expensive instrumentation.

Acknowledgement. This work was supported by the Slovak Research and Development Agency under the contracts Nos. LPP-0038-09 and by the Slovak Grant Agency VEGA grants Nos. 2/0162/10, 2/0217/10.