Automated protein crystallization application on Hamilton's MICROLAB-STAR pipetting station

With the significant development of the industry and academia in the field of proteomics, people's attention has turned to the study of proteins and their three-dimensional structure and function. This includes the use of protein crystallization to determine protein structure. Protein crystallization is a multifactorial process that depends on the interaction of many independent parameters. These parameters include: pH, temperature, protein concentration, crystallizer concentration, presence or absence of impurities or additives, and their properties. For parallel crystallization optimization experiments, Hamilton has developed the MICROLAB® STAR Protein Crystallization Workstation. The workstation can be used for both primary and secondary screening. It can be either a hanging drop method or a drop method. It can be used with custom reagents or commercial kits. It can be used in 96-well or 384-well crystals. board.

Instruments and materials
MICROLAB-STAR Protein Crystallization Workstation (P/N 187007)
MICROLAB-STAR 8-channel pipetting system, sample loading
MICROLAB-SWAP microplate operation manipulator special reagent carrier crystal special experimental equipment carrier anti-volatile cover microplate storage chamber cutting (storage unit)
Linbro board carrier (optional, for hanging drop method)

Kit Screening Kit (P/N 187008)
Falcon tube carrier deep hole carrier extended bar code reader (optional)
Wash station for standard steel needles (optional)

Refine the screening package (P/N 187009)
Special reagent carrier extended bar code reader for standard steel needle washing station for large volume steel needle washing station (optional)
(The filter package does not include the worksheet)

Operating procedures
Protein crystallization involves two main steps: one is to transfer the crystallization solvent into the main pore of the crystallized plate, and the other is to suspend the protein solution in the crystallization solvent in the adjacent pores of the crystal plate. MICROLAB® STAR Protein Crystallization Workstation (Figure 1)

Customized crystallization solvents can be formulated using the stored solution and the solvents in the available screening kits.

The MICROLAB-STAR Protein Crystallization Workstation is also available with an optional kit for screening kits. A refinement screening package can also be selected for refinement screening. Before starting the crystallization process, MICROLAB-SWAP will check the microplate chamber for sufficient microplates to allow the program to operate under unmanned conditions and, if necessary, read the microplate barcode. Each plate will be placed with the anti-volatile lid onto the crystallized frame carrying the microplate and lid. The lid of the crystal plate and the reagent plate was opened, and up to 150 μl of the crystallization solvent was added to the crystal plate. After the solvent is added to the hole of the crystal plate, different volume of protein solution (usually 0.5-1.0 μl) and an equal volume of crystal solvent are taken up and added to the pores of the crystal plate. This step does not require a mixing process, precluding the possibility that the protein and solvent will not mix well in the pores. The crystallized plates are processed column by column when the protein is added. Immediately after adding a column of MICROLAB-SWAP, cover the anti-volatile cover.

Kit Screening <br> When using the screening kit, the crystallization solvent must first be dispensed from the solvent tube into a 96-deep well plate and immediately capped with a vapor-proof lid to ensure that the concentration of the prepared solution does not change. The pipetting and transfer operations after this step are completed are the same as described above. The kit screening kit is suitable for commercial kits.

Refinement Screening <br> The formulation of the crystallization solvent required for the refinement screening is determined by the worksheet. The mixing method for storing reagents is defined in the worksheet. The MICROLAB-Vector software searches for incomplete worksheets or missing microplates and eliminates them before running. The workstation will also check if there are enough reagents to complete the worksheet. These checks ensure a reliable and decentralized automated refinement of the screening setup process. The pipetting and transfer operations after this step are completed are the same as described above. Refinement screening requires the use of a refinement screening package. MICROLAB-STAR can use both disposable tips and reusable stainless steel needles to remove crystallization solvents and other reagents. The step of removing the protein is done with a disposable tip. Thanks to the patented CO-RE technology, the positioning accuracy of STAR tips can reach 0.1 mm in all axial directions, far beyond the small holes to be added to the sinking microplate (the pitch is the same as the 1536 well plate). The precision required in ).

result

Validation was performed on a protein crystallization workstation using test proteins and commercial kits under the same conditions as manual procedures. Crystal growth occurred 4 days after the primary screening conditions (Fig. 3a). Refinement screening under optimized conditions yielded fewer but larger crystals (Fig. 3b).
The accuracy of the 0.5 μl + 0.5 μl protein/solvent pipetting step was 7% by the weighing method. Measurement by weighing,
the deep well plates after 18 hours at room temperature with a liquid head loss of less than 0.4%, without loss in the deep-well plate lid liquid reached 8.5%.

Flux If a mixing process is not required, the Protein Crystallization Workstation can process a 96-well Greiner Crystal Quick Plate in 20 minutes. In the refinement screening experiment, depending on the complexity of the crystallization solvent mixture, it takes 60 to 90 minutes per plate to be processed. Using the kit screening kit, the Protein Crystallization Workstation can perform 16 commercial screenings with deep well plates in 2 hours and 40 minutes. Using a refinement screening package, 16 knotted crystal plates can be processed in 24 hours.

Discussion <br>Hamilton has developed a protein crystallization workstation for fully automated protein crystallization with highest reliability and positioning and pipetting accuracy. Once the program has completed inspection of the microplates, reagents, and worksheets, the workstation will no longer require user intervention.

Features and Benefits Through the column-by-column treatment of porous plates, the experimental design of the crystallization conditions is flexible. The flexible porous plate and the flexible use of the experimental equipment can be used alternately with disposable tips and reusable steel needles. Cost-saving automatic system inspection ensures maximum reliability. And can be separated from people

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