Experiences with the use of the Microbatch Under Oil Method of Crystallisation Comparison of...
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Experiences with the use of the Microbatch Under Oil Method of Crystallisation I-dom ain 2 VD 1 l+ 1 l MB 1 l+ 1 l MB 0.5 l +0.5 l MB 0.3 l +0.3 l MB 0.2 l +0.2 l Totalno.of conditions 5 10 10 10 10 N o.of unique conditions* 0 7 6 6 6 N o.of com m on conditions* 5** 3 4 4 4 D rop Size Tim e taken for drops to dry out VD :1 lprotein+ 1 l reservoir > 1 year M B:1 lprotein+ 1 l reservoir 40 days MB:0.5 l+ 0.5 l 27 days MB:0.3 l+ 0.3 l 20 days (m ostdrops dried out) MB:0.2 l+ 0.2 l 20 days (alldrops dried out) P rotein Conditions unique to VD Conditions unique to M B C onditions in com m on C onditions screened I-dom ain 1 8 5 3 48 Cytokine 3 0 0 48 I-dom ain 2 2 7 3 48 FA B 43 7 4 384 Ligase 3 9 0 576 K inase 1 activated 10 8 11 96 K inase 2 not activated 4 0 0 192 K inase 3 not activated 30 54 29 192 Sub Total 103 95 50 1584 Comparison of different drop volumes suitable for Microbatch crystallisation: * versus VD 1l+1l, **VD versus all MB Common conditions between MB 1l+1l and MB 0.5l+0.5l (or 0.3l, 0.2l): 6 Drying out of drops- Time verses Drop volume: Trays were stored at room temperature in a cupboard. This investigation gives us a general idea of how long an experiment will last. The smallest volumes dry out very quickly. This may prevent discovery of new crystallisation conditions. Comparison of Microbatch and Vapour diffusion for screening. Vapour diffusion (VD) experiments were done with drop ratios of 1+1 or 2+2 L. Microbatch (MB) experiments were generally 0.5+0.5 L or less. All screens are commercially available (i.e. Hampton Research, Jena Bioscience, Emerald biostructure). Introduction : The Microbatch under oil method of crystallisation is a good way of screening many crystallisation conditions using a very small amount of protein. We have screened a variety of different proteins using the Oryx robot from Douglas Instruments (microbatch method) and the Cyberlab C200 robot from Gilson (hanging drop method of vapour diffusion). Our aim was to determine the relative success rate of microbatch versus vapour diffusion, the size of the drops that could be used for microbatch and the best way of storing these experiments. We have also started investigating using standard 96 and 384 well trays for storing of stock solutions and screening experiments. We have tested various methods of storage for prolonging the lifetime of the experiments with 0.2l+0.2l and 0.3l+0.3l drop volumes. MB drop volumes as small as 0.2 + 0.2 L are equally successful compared to larger volumes Only 6 out of a total of 10 conditions are common between MB 1+1 L and the smaller drop volumes. There are a similar number of common conditions with VD for any of the MB volumes. For this particular protein, MB gives more unique conditions than VD and all of the VD conditions were also found using MB. Adding a reservoir of water to a particular box increases the lifetime of the experiments by approx. 25 days. As expected, polypropylene boxes are more effective than polystyrene. Nafeesa Noordeen & Sandra Cowan-Jacob, Novartis Pharma AG, Basel, Switzerland. Methods: The oil-dropping-on method is used in preference to dispensing drops under oil. We find that the former method gives more centred drops when dispensing very small drop volumes. 15L of paraffin oil was dispensed on top of each drop and then the whole tray was covered with about 4 ml of Al’s oil (50:50 Paraffin/Silicon) to let Both methods are equally successful overall in finding initial crystallisation conditions. However, different proteins can be more successful in one or the other methods. Hence, use both methods whenever possible. Conclusions: • Microbatch and Vapour diffusion are equally successful in screening for initial crystallisation conditions. • It is possible to use small volumes (0.2l+0.2l) for MB screening as it gives a similar number of crystallisation conditions as larger MB volumes. •Storage in polypropylene boxes is most effective in prolonging the lifetime of small volume experiments (but only to a month ). Adding a reservoir of water to any type of storage box increases the lifetime by about 25 - 30 days. •The ability of the robot to perform 96 well experiments means that the method is more automated and requires less operator time per 48 wells.
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Transcript of Experiences with the use of the Microbatch Under Oil Method of Crystallisation Comparison of...
Method ofstorage
Volume of dropsTime taken for dropsto dry out
1.Polypropylenebox
0.l protein+0.3lbuffer
29 days
2.Polypropelenebox + H2Oreservoir
0.3l protein+0.3lbuffer
>60 days – not driedout as yet
3.Polystyrenebox
0.2l H20 + 0.2lbuffer
0.2l protein+0.2lbuffer
18 days
22 days
4.Polystyrenebox + H2Oreservoir
0.2l H20 + 0.2lbuffer
0.2l protein+0.2lbuffer
45 days
44 days.
5.Polystyrenebox sealed withtape
0.2l H20 + 0.2lbuffer
0.2l protein+0.2lbuffer
19 days
24 days
6.Cryo box 0.2l H20 + 0.2lbuffer
0.2l protein+0.2lbuffer
19 days
19 days
7.Cryo box +H2O reservoir
0.2l H20 + 0.2lbuffer
0.2l protein+0.2lbuffer
44 days
>38 days -not driedout as yet.
8.Polystyrenebox2
1l H20+ 1l H20
0.5l H20+ 0.5l H20 0.2l H20 + 0.2l H2O
>51 days – not driedout as yet.44 days19 days
Experiences with the use of the Microbatch Under Oil Method of Crystallisation
I-domain 2 VD1l + 1l
MB1l + 1l
MB0.5l+0.5l
MB0.3l+0.3l
MB0.2l+0.2l
Total no. ofconditions 5 10 10 10 10
No. ofuniqueconditions*
0 7 6 6 6
No. ofcommonconditions*
5** 3 4 4 4
Drop Size Time taken for drops todry out
VD: 1 l protein+ 1lreservoir
> 1 year
MB: 1 l protein+ 1lreservoir
40 days
MB: 0.5 l + 0.5 l 27 days
MB: 0.3 l + 0.3 l 20 days (most dropsdried out)
MB: 0.2 l + 0.2 l 20 days (all dropsdried out)
ProteinConditions
unique to VDConditions
unique to MBConditions in
commonConditions
screened
I-domain 1 8 5 3 48
Cytokine 3 0 0 48
I-domain 2 2 7 3 48
FAB 43 7 4 384
Ligase 3 9 0 576Kinase 1activated 10 8 11 96Kinase 2 notactivated 4 0 0 192Kinase 3 notactivated 30 54 29 192Sub Total
103 95 50 1584
Comparison of different drop volumes suitable for Microbatch crystallisation:
* versus VD 1l+1l, **VD versus all MBCommon conditions between MB 1l+1l and MB 0.5l+0.5l (or 0.3l, 0.2l): 6
Drying out of drops- Time verses Drop volume:
Trays were stored at room temperature in a cupboard.
This investigation gives us a general idea of how long an experiment will last. The smallest volumes dry out very quickly. This may prevent discovery of new crystallisation conditions.
Comparison of Microbatch and Vapour diffusion for screening.Vapour diffusion (VD) experiments were done with drop ratios of 1+1 or 2+2 L. Microbatch (MB) experiments were generally 0.5+0.5 L or less.All screens are commercially available (i.e. Hampton Research, Jena Bioscience, Emerald biostructure).
Introduction: The Microbatch under oil method of crystallisation is a good way of screening many crystallisation conditions using a very small amount of protein.
We have screened a variety of different proteins using the Oryx robot from Douglas Instruments (microbatch method) and the Cyberlab C200 robot from Gilson (hanging drop method of vapour diffusion). Our aim was to determine the relative success rate of microbatch versus vapour diffusion, the size of the drops that could be used for microbatch and the best way of storing these experiments. We have also started investigating using standard 96 and 384 well trays for storing of stock solutions and screening experiments.
We have tested various methods of storage for prolonging the lifetime of the experiments with 0.2l+0.2l and 0.3l+0.3l drop volumes.
MB drop volumes as small as 0.2 + 0.2 L are equally successful compared to larger volumes
Only 6 out of a total of 10 conditions are common between MB 1+1 L and the smaller drop volumes.
There are a similar number of common conditions with VD for any of the MB volumes.
For this particular protein, MB gives more unique conditions than VD and all of the VD conditions were also found using MB.
Adding a reservoir of water to a particular box increases the lifetime of the experiments by approx. 25 days. As expected, polypropylene boxes are more effective than polystyrene.
Nafeesa Noordeen & Sandra Cowan-Jacob,Novartis Pharma AG, Basel, Switzerland.
Methods:The oil-dropping-on method is used in preference to dispensing drops under oil. We find that the former method gives more centred drops when dispensing very small drop volumes. 15L of paraffin oil was dispensed on top of each drop and then the whole tray was covered with about 4 ml of Al’s oil (50:50 Paraffin/Silicon) to let some evaporation occur.
Both methods are equally successful overall in finding initial crystallisation conditions.
However, different proteins can be more successful in one or the other methods.
Hence, use both methods whenever possible.
Conclusions:
• Microbatch and Vapour diffusion are equally successful in screening for initial crystallisation conditions.
• It is possible to use small volumes (0.2l+0.2l) for MB screening as it gives a similar number of crystallisation conditions as larger MB volumes.
•Storage in polypropylene boxes is most effective in prolonging the lifetime of small volume experiments (but only to a month ). Adding a reservoir of water to any type of storage box increases the lifetime by about 25 - 30 days.
•The ability of the robot to perform 96 well experiments means that the method is more automated and requires less operator time per 48 wells.
