Microbatch

Microbatch crystallization has recently become a popular choice, for a number of reasons. First, microbatch generally uses much less protein and reagents than vapor diffusion. In addition, microbatch is much more amenable to using robotics, and there are a number of proteomics groups that have turned to microbatch for their high-throughput programs. Lastly, microbatch will often yield different crystallization conditions than vapor diffusion (Baldock et al., 1996), so the two methods can be complementary to each other. 

Baldock P, Mills V, Stewart PS. 1996. A comparison of microbatch and vapor diffusion for initial screening of crystallization conditions. J Cryst Growth 168 170-174. 

B. Zheng, J.D. Tice, L.S. Roach, and R.F. Ismagilov A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with on-Chip X-Ray Diffraction. Angew. Chem. Int. Ed. 43, 2508 (2004). 

The first semi-high-throughput automated system to dispense crystallization trials of less than 1 jl1 was designed in 1990 to deliver batch trials imder oil (Chayen et ah, 1990). The method was named microbatch to define a microscale batch experiment. It was designed to obtain maximum information on the molecule to be crystallized while using minimal amounts of sample. In order to prevent the evaporation of such small volumes, the trials are dispensed and incubated under low density (0.87 g/cm ) paraffin oil (Fig. 3.2). The crystallization drops remain under the oil since the aqueous drops are denser than the paraffin oil. 

Microbatch can be performed either manually or automatically (Chayen et ah, 1992). It is the simplest crystallization method and therefore can be easily performed in high-throughput trials. Current robots can dispense microbatch trials down to 1 nl volumes. Depending on the type of oils used to cover the trials, this technique can be harnessed for both screening and optimization experiments. 

Figure 3.2 Dispensing of a microbatch trial under oil. The dashed circle represents the initial position of the crystallization drop at the time of dispensing. The grey circle represents the final position of the drop after it had made its way to the bottom of the well (due to its higher density compared to that of the oil). Modified from Chayen (1997). The role of oil in macromolecular crystallization. Structure 5, 1259-1274, Copyright Elsevier.

A modification of the original microbatch method provides a means of simultaneously retaining the... 

An increasing number of membrane proteins, in a variety of different detergents, have been crystallized in microbatch under oil. Some of these had failed to crystallize by all methods other than microbatch. Dispensing is quick and simple, even when performed manually, and the drops in oil do not spread out as they do in vapour diffusion over the siliconized coverslips (Chayen, 2006). Using robots thousands of microbatch trials can be dispensed in high-throughput mode in nanolitre volumes. 

The protocol for setting up microbatch experiments containing membrane proteins is identical to that described in Protocols 3.1 and 3.2. 

Harvesting crystals from microbatch is slightly more difficult than harvesting from coverslips or from standard sitting drops (Protocol 3.3). However, after... 

Although microbatch is the simplest method of crystallization, it is a relatively new technique and many experimenters still prefer to use vapour diffusion which has been around and has worked well for over 40 years. Hence, there has also been major development in automating and scaling down the quantities of sample using the popular vapour diffusion method (both sitting and hanging drops). An increasing variety of robots are available commercially. 

Protocol 3.1 Setting up a screen in a microbatch experiment manually... 

Paraffin oil (Hampton Research or Molecular Dimensions) Silicone oil (Hampton Research or Molecular Dimensions) Microbatch plates (e.g. Nunc, Terazaki, Douglas Instruments)... 

Pipette or pour 6 ml of paraffin oil into a microbatch plate. The oil will spread over the plate and cover the wells. 

Microbatch plates Protein solution Crystallization reagents Paraffin oil... 

Protocol 3.3 Two alternative ways of harvesting crystals from microbatch... 

An alternative to dilution in microbatch is to approach the optimization strategy from the opposite... 

Protocol 3.7 Induction and subsequent arrest of nucleation in microbatch ... 

Microbatch plates 3. Top up the oil (at different times for the different trays)... 

Protein solutions 4. Continue to incubate as standard microbatch trials. 

Method 1. Set up several oystallization plates containing microbatch trials under a layer of paraffin oil so that the oil just covers the trials (Fig. 3.5a). time intervals after setting up the trials. 

Figure 3.5 Controlled evaporation of crystallization drops in microbatch, (a) Trials incubated under a thin layer of oil that allows concentration via evaporation, thus leading to nudeation. (b) Arrest of evaporation/concentration by addition of oil to produce a thicker layer above the trials. Modified from Chayen and Saridakis (2002), Acta Cryst. D 58, 921-927, with permission from the lUCr.

Protocol 3.10 Dispensing microbatch crystallization trials in gels... 

Fill a crystallization tray with paraffin oil as done for standard microbatch trials (by hand or robot). 

Han/esting crystals from the gelled drops is done in the same way as from standard microbatch trials since the gel is quite tenuous. 

Chayen, N. E. (1998). Comparative studies of protein crystallization by vapour-diffusion and microbatch techniques. Acta Crystallogr. D 54, 8-15. 

Chayen, N. E., Stewart, P. D. S. and Blow, D. M. (1992). Microbatch crystallization under oil - a new technique allowing many smaU-volume crystallization trials. J. Crystal Growth 122,176-180. 

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