Wizard: Precipitant Synergy Crystallization Screen
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Discover more protein crystallization hits with this unique screen by employing synergistic effects of mechanistically distinct precipitating agents (salts, organic solvents, polyethylene glycols, additives, pH). The Wizard: Precipitant Synergy crystallization consists of 192 formulations, to be used for initial protein crystallization screening and crystallization optimization. The application of this screen is advised in particular for protein targets that have failed to yield hits with traditional random sparse matrix crystallization approaches.
96-Well Block Plate formulations are 1.0 ml each in a 96 deep-well matrix block plate sealed with a sterile reusable mat. Tube sets are 10 ml volumes per tube. All formulations are prepared with ASTM type-1 water and high-purity chemicals, 0.2 µm filtered in sterile packaging.
The Wizard: Precipitant Synergy protein crystallization screen targets all types of soluble protein and protein complexes. Tests have shown that this screen can triple the number of unique crystals when compared with other screens (Mayeed et al., 2003). Each unique formulation is made available in three different concentrations, increasing the coverage of crystallization space. These related formulations are positioned in neighboring wells or tubes, simplifying the analysis of crystallization behavior.
Each Wizard: Precipitant Synergy formulation contains a selected combination of at least two distinct precipitating agents, allowing the assessment of crystallization factors in five independent dimensions:
(i) 8 different salts [affects the water activity]
(ii) 4 different organic solvents [modulates solvent dielectricity]
(iii) 4 different poly ethylene glycols [causes molecular crowding]
(iv) 9 different additives [mediates crystal packing]
(v) 5 different pH values covering a pH range from 4.5 to 8.5 [alters protein surface charge]
Sampling of the resulting five dimensional crystallization space is balanced by the selected pH and assures good coverage of the binary precipitant mixtures.
Each of the 64 base formulations contain precipitants at high concentration, close to their maximum solubility, in order to reach or exceed the protein’s precipitation point. Corresponding formulations with precipitant concentrations at 67%, and 33%, but same buffer concentration ensures finer grid coverage of crystallization space and systematic crystallization.
192 formulations, 1.7 mL volumes in two 96 well block plates.
Application: Wizard Precipitant Synergy formulations in blocks are particularly well suited for high-throughput protein crystallization in 96 well standard formats, using multichannel pipettors or robotic dispensation instrumentation.
Recommended Use Depending on the type of crystallization tray and employed reservoir volume, a single Wizard Precipitant Synergy protein crystallization kit provides formulations for ca. 12 to 24 96-well based crystallization plates (assuming reservoir well volumes of 160 or 80 uL).
Approximate total number of crystallization setups: 1,152 – 2,304 corresponding to six or twelve unique 192 formulation crystallization trials.
Dimensions: Well positions of deep well block plates conform to well center positional requirements for microplates (ANSI/SBS 4-2004).
Well shape: round
Well diameter: 7mm
Well depth: 35mm
Well bottom shape: round
Block dimensions: 128 x 86 x 42 mm
Formulations arrangement: 64 base Precipitant Synergy formulations are arranged sequentially in each row, with every third well filled with a unique set of reagents. Three neighboring wells in rows each contain a base formulation consisting of 100%, 67% and 33% of precipitation reagents and constant buffer concentrations of 100 mM.
The Precipitant Synergy Protein Crystallization Screen Kit contains 192 distinct Precipitant Synergy protein crystallization formulations.
Application: Precipitant Synergy formulations are used to prepare protein crystallization experiments in a variety of experiment formats: including vapor diffusion or batch crystallization, sitting or hanging drop configurations.
Recommended Use: Primary protein crystallization trials, particularly difficult-to-crystallize proteins.
Primary screening of one target protein using a total of 192 different crystallization formulations. Simple coverage of 768 distinct crystallization conditions can be achieved by setting up crystallization experiments employing two protein concentrations (i.e. 20 and 10 mg/mL) and incubation at 4ºC and room temperature.
Long Term Storage: Room temperature
Use / Stability: Kit formulations are stable for one year from date of receipt
Handling: Avoid freezing. Avoid Temperatures above 37ºC. Protect from light. Once opened, reseal with tape to reduce evaporation.
Validation: Crystallization tests showed that the use of Wizard Precipitant Synergy formulations “…significantly enhanced both the probability of crystallization as well as the quality of optimized crystals”. Tests with several difficult to crystallize proteins showed a significant two to three-fold increase in the overall probability of crystallization (Mayeed et al., 2003).
Literature References Majeed, S., Ofek, G., Belachew, A., Huang, C.C., Zhou, T., Kwong, P.D. (2003) Enhancing protein crystallization through precipitant synergy. Structure, 11: 1-20
US patent: 7,300,520