Non-robotic high-throughput setup for manual assembly of nanolitre vapour-diffusion protein crystallization screens

Nanolitre-sized drops are characteristic of high-throughput protein crystallization screening. Traditionally, reliable nanolitre drop dispensing has required the use of robotics. This work describes the design and development of 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 together with simple tools available in standard laboratories. The method saves precious protein material without sacrificing the effectiveness of the screening process. To verify the approach, two monoclonal antibody Fab fragments were crystallized alone and in a complex with tau peptide antigens in 0.2-0.5 µl drops. Crystals grown directly from the screen conditions in sitting drops on 96-well plates diffracted up to 1.6 Å resolution on a synchrotron source. The results proved that successful crystallization in nanolitre high-throughput format is affordable even in the absence of expensive robotic instrumentation.