Validation of a high-throughput setup for manual assembly of nanolitre vapour-diffusion protein crystallization screens

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 SE, Grosslingova 45, 811 09 Bratislava, Slovakia

Nanolitre-sized drops are characteristic of high-throughput protein crystallization screening. Traditionally, reliable nanolitre drop dispensing has required the use of robotics. We have developed a protocol for the reproducible manual assembly of nanolitre-sized protein vapour-diffusion crystallization trials in a 96/192-drop format. The protocol exploits the repetitive-pipetting mode of handheld motorized pipettes and saves precious protein material without sacrificing the effectiveness of the screening process. The manual dispensing of solutions with variable viscosity in the range of 0.2 – 0.5 µl by a motorized multichannel pipette was comparably reliable as dispensing by robotics [1] or by a handheld nanoject pipettor [2]. We have tested the repeatability and reproducibility of proposed method by repeating successful crystallization trials of an antibody Fab fragment in sitting drops on 96-well plates with variable precipitant volume and comparing motorized and manual mode of pipetting. We were able to produce crystals repeatedly in 0.5 and 0.3 µl drops and in a 0.2 µl drop assembled by a manual pipette. Summarizing, developed protocol allows for reproducible manual formation of nanolitre protein crystallization drops in high-throughput format. We believe that the method could be useful especially for small crystallographic groups since it needs no additional expensive instrumentation [3].

This work was supported by the Slovak Research and Development Agency under the contract No. LPP-0038-09 and by the Slovak Grant Agency VEGA grant No. 2/0163/13.

1. Walter, T. S., Diprose, J., Brown, J., Pickford, M., Owens, R. J., Stuart, D. I. & Harlos, K. (2003). Journal of Applied Crystallography 36, 308-314

2. Cherezov, V. & Caffrey, M. (2005). Journal of Applied Crystallography 38, 398-400

3. Skrabana, R., Cehlar, O. & Novak, M. (2012). J. Appl. Cryst. 45, 1061–1065.