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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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Chemical structures of cubic phase-forming lipids. (A) 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4), (B) 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4)
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Fig1: Chemical structures of cubic phase-forming lipids. (A) 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4), (B) 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4)

Mentions: We employed two isoprenoid chained lipid species, 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4) and 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4), as cubic phase forming lipids. β-XylOC16+4 and EROCOC17+4 were the same materials described in the previous papers [9, 10, 12], and the chemical structures are shown in Fig. 1. Lysozyme (cat: L-6876) and Bacteriorhodopshin from Halobacterium salinarum (cat: B0184) were obtained from Sigma (St. Louis, Mo. USA).Fig. 1


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)

Chemical structures of cubic phase-forming lipids. (A) 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4), (B) 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Chemical structures of cubic phase-forming lipids. (A) 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4), (B) 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4)
Mentions: We employed two isoprenoid chained lipid species, 1-O-(3,7,11,15-tetramethylhexadecyl)-β-d-xylopyranoside (β-XylOC16+4) and 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCOC17+4), as cubic phase forming lipids. β-XylOC16+4 and EROCOC17+4 were the same materials described in the previous papers [9, 10, 12], and the chemical structures are shown in Fig. 1. Lysozyme (cat: L-6876) and Bacteriorhodopshin from Halobacterium salinarum (cat: B0184) were obtained from Sigma (St. Louis, Mo. USA).Fig. 1

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