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Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering.

Oberthuer D, Melero-García E, Dierks K, Meyer A, Betzel C, Garcia-Caballero A, Gavira JA - PLoS ONE (2012)

Bottom Line: Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes.Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation.Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of infection and inflammation, University of Hamburg, Hamburg, Germany.

ABSTRACT
In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

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Related in: MedlinePlus

Comparison of DLS measurements in thin capillaries and a quartz cuvette.A) It shows an overlay plot of the radius distribution of glucose isomerase in capillaries and a quartz cuvette; B) It shows an overlay of the auto correlation function from which the radius distribution were derived; C) Picture showing the focus of the laser inside a 0.1 mm capillary and the scattering associated to the capillary-walls, which does not affect the measurements.
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pone-0033545-g001: Comparison of DLS measurements in thin capillaries and a quartz cuvette.A) It shows an overlay plot of the radius distribution of glucose isomerase in capillaries and a quartz cuvette; B) It shows an overlay of the auto correlation function from which the radius distribution were derived; C) Picture showing the focus of the laser inside a 0.1 mm capillary and the scattering associated to the capillary-walls, which does not affect the measurements.

Mentions: The volume of solution analysed by the DLS system (crossing space of laser and detector) has been previously estimated at around 1 pL [14]. Such volume is equivalent to a sphere of approximately 12.5 micrometres in diameter and should not be neglected for measurements in 100 micrometres capillaries that, on the other hand, are the most widely used because of its small sample consumption (approximately 400 nL). Therefore, initially it was necessary to establish whether it is possible to obtain a clean DLS signal similar to that obtained in quartz cuvettes without any special distortion due to the geometry of the capillary or the plastic walls of the GCB-D. Different sets of DLS data of a protein solution were taken in a quartz cuvette and capillaries of varying diameters for comparative purposes. Three capillaries of 0.1, 0.2, of 0.3 mm plus an extra capillary of 0.3 mm with agarose were filled with a solution of glucose isomerase in its buffer and subsequently punctured in GCB-D’s filled with 3 M ammonium sulphate. A DLS measurement was taken at 30 mm from the open end of the capillaries to make sure that the precipitant had not reached that position. Then, a total of 15 DLS measurements at 2 seconds interval were taken for each capillary and the quartz cuvette. The results of the size distributions obtained from the DLS signal are shown in Figure 1.


Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering.

Oberthuer D, Melero-García E, Dierks K, Meyer A, Betzel C, Garcia-Caballero A, Gavira JA - PLoS ONE (2012)

Comparison of DLS measurements in thin capillaries and a quartz cuvette.A) It shows an overlay plot of the radius distribution of glucose isomerase in capillaries and a quartz cuvette; B) It shows an overlay of the auto correlation function from which the radius distribution were derived; C) Picture showing the focus of the laser inside a 0.1 mm capillary and the scattering associated to the capillary-walls, which does not affect the measurements.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366972&req=5

pone-0033545-g001: Comparison of DLS measurements in thin capillaries and a quartz cuvette.A) It shows an overlay plot of the radius distribution of glucose isomerase in capillaries and a quartz cuvette; B) It shows an overlay of the auto correlation function from which the radius distribution were derived; C) Picture showing the focus of the laser inside a 0.1 mm capillary and the scattering associated to the capillary-walls, which does not affect the measurements.
Mentions: The volume of solution analysed by the DLS system (crossing space of laser and detector) has been previously estimated at around 1 pL [14]. Such volume is equivalent to a sphere of approximately 12.5 micrometres in diameter and should not be neglected for measurements in 100 micrometres capillaries that, on the other hand, are the most widely used because of its small sample consumption (approximately 400 nL). Therefore, initially it was necessary to establish whether it is possible to obtain a clean DLS signal similar to that obtained in quartz cuvettes without any special distortion due to the geometry of the capillary or the plastic walls of the GCB-D. Different sets of DLS data of a protein solution were taken in a quartz cuvette and capillaries of varying diameters for comparative purposes. Three capillaries of 0.1, 0.2, of 0.3 mm plus an extra capillary of 0.3 mm with agarose were filled with a solution of glucose isomerase in its buffer and subsequently punctured in GCB-D’s filled with 3 M ammonium sulphate. A DLS measurement was taken at 30 mm from the open end of the capillaries to make sure that the precipitant had not reached that position. Then, a total of 15 DLS measurements at 2 seconds interval were taken for each capillary and the quartz cuvette. The results of the size distributions obtained from the DLS signal are shown in Figure 1.

Bottom Line: Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes.Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation.Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of infection and inflammation, University of Hamburg, Hamburg, Germany.

ABSTRACT
In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.

Show MeSH
Related in: MedlinePlus