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A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser.

Hato M, Hosaka T, Tanabe H, Kitsunai T, Yokoyama S - J. Struct. Funct. Genomics (2014)

Bottom Line: The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing.Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well.The system was useful for crystallizing membrane and soluble proteins in meso.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan, hato-m@gsc.riken.jp.

ABSTRACT
A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.

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Protein crystals grown by using the new manual dispensing system. Bacteriorhodopsin/β-XylOC16+4 (A), lysozyme/EROCOC17+4 (B), proteorhodpsin/EROCOC17+4 (C), and human hormone receptor/EROCOC17+4 (D). All crystallizations were performed at 20 °C, except for that of the lysozyme/EROCOC17+4 system, which was performed at 4 °C
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Fig7: Protein crystals grown by using the new manual dispensing system. Bacteriorhodopsin/β-XylOC16+4 (A), lysozyme/EROCOC17+4 (B), proteorhodpsin/EROCOC17+4 (C), and human hormone receptor/EROCOC17+4 (D). All crystallizations were performed at 20 °C, except for that of the lysozyme/EROCOC17+4 system, which was performed at 4 °C

Mentions: To further demonstrate the performance of the new manual dispensing system, we performed in meso crystallization trials of several membrane proteins, as well as water-soluble lysozyme, using β-XylOC16+4 and EROCOC17+4 as matrix lipids for crystallization. We followed the standard in meso crystallization procedure [5], with 50 nl protein-laden mesophases and 1–0.8 μl crystallization solutions. All crystallizations were performed at 20 °C except for the lysozyme/EROCOC17+4 system, which was performed at 4 °C. The protein crystals thus obtained are shown in Fig. 7.Fig. 7


A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser.

Hato M, Hosaka T, Tanabe H, Kitsunai T, Yokoyama S - J. Struct. Funct. Genomics (2014)

Protein crystals grown by using the new manual dispensing system. Bacteriorhodopsin/β-XylOC16+4 (A), lysozyme/EROCOC17+4 (B), proteorhodpsin/EROCOC17+4 (C), and human hormone receptor/EROCOC17+4 (D). All crystallizations were performed at 20 °C, except for that of the lysozyme/EROCOC17+4 system, which was performed at 4 °C
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125823&req=5

Fig7: Protein crystals grown by using the new manual dispensing system. Bacteriorhodopsin/β-XylOC16+4 (A), lysozyme/EROCOC17+4 (B), proteorhodpsin/EROCOC17+4 (C), and human hormone receptor/EROCOC17+4 (D). All crystallizations were performed at 20 °C, except for that of the lysozyme/EROCOC17+4 system, which was performed at 4 °C
Mentions: To further demonstrate the performance of the new manual dispensing system, we performed in meso crystallization trials of several membrane proteins, as well as water-soluble lysozyme, using β-XylOC16+4 and EROCOC17+4 as matrix lipids for crystallization. We followed the standard in meso crystallization procedure [5], with 50 nl protein-laden mesophases and 1–0.8 μl crystallization solutions. All crystallizations were performed at 20 °C except for the lysozyme/EROCOC17+4 system, which was performed at 4 °C. The protein crystals thus obtained are shown in Fig. 7.Fig. 7

Bottom Line: The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing.Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well.The system was useful for crystallizing membrane and soluble proteins in meso.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan, hato-m@gsc.riken.jp.

ABSTRACT
A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.

Show MeSH
Related in: MedlinePlus