8.4 Crystallographic methods
8.4.1 Sitting drop
The sitting drop method[188] was used in 96 well format during screening for initial crystal-lization conditions with commercially available prefilled screens. Therefore, 100µl of each 96 screen solution was pipetted into a 96 well plate using a multi pipette. Afterwards, this 96 well plate was used with a Cartesian roboter, which pipetted 100, 200 and 400 nl of the reservoir condition into the pre- pipetted 200 nl protein drop. The plates were sealed with crystal clear adhesive foil and transferred into a temperated18◦Ccrystallization closet.
Figure 8.2: Sitting drop vapour diffusion method. Arrow represents transport of water to the reservoir solution containing a higher precipitant concentration (McPherson, 1998[188]).
Figure 8.3: Hanging drop vapour diffusion method. Arrows represent the netto transport of water. (McPherson, 1998[188]).
8.4.3 Hyper quenching
Hyper quenching was tested to improve crystal freezing.[178,179] The idea of hyper quenching is to increase the cooling rate for the crystal after fishing during freezing in liquid nitrogen.
Warkentinet al.[178,179]found that the temperature of the air layer above liquid nitrogen is below 0◦Cand leads to a slower cooling rate of the fished crystal when dipping the crystal into liquid nitrogen. A very simple design was used to remove the cool air layer above the liquid nitrogen.
The dewar containing liquid nitrogen was filled up to the lip and a dry air stream was blown from the side of the dewar to remove the cool air layer. Crystals were flash frozen by increased dipping speed. Significant differences between freezing with or without hyper quenching could not be detected, however, no decline in the crystal quality could be detected, too.
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