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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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Related in: MedlinePlus

The new microsyringe-based system for dispensing. A The new removable needle consisting of an Ito microsyringe needle (a) and a fitting ring (b). B A schematic diagram of the 96-hole polyester dual adhesive sheet (a) and the 96-hole spacer plate (b). Dimensions are given in millimeters. C The lipid-protein homogenization device with the monolithic stainless steel coupler
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Fig3: The new microsyringe-based system for dispensing. A The new removable needle consisting of an Ito microsyringe needle (a) and a fitting ring (b). B A schematic diagram of the 96-hole polyester dual adhesive sheet (a) and the 96-hole spacer plate (b). Dimensions are given in millimeters. C The lipid-protein homogenization device with the monolithic stainless steel coupler

Mentions: Homogenization of the lipid/water or lipid/protein buffer mixtures was performed at room temperatures (20–23 °C) by using a device consisting of a pair of Ito MS-GAN025 (250 μl) microsyringes with a homebuilt monolithic stainless steel coupler (Fig. 3C). The detailed structure and the homogenization procedures of this device were described in a previous paper [11], where the achievement of molecular homogeneity of lipid/water mixtures was confirmed by a small angle X-ray scattering measurements [10–12]. Though the homogenization procedures were essentially the same as those in the standard in meso protocol [5], there was one major difference in the lipid-loading step. The lipids used in this work are viscous waxy material over a temperature range from 0 to at lest ~80 °C [10, 12]. Thus, the lipids (usually 10–30 mg) were loaded into the open end of the microsyringe at room temperatures (without heating) by using a homebuilt stainless steal microspatula followed by an immediate homogenization with water or a protein buffer. In loading solid monoacylglycerols, e.g., monoolein, heating of the lipid at ~40 °C before or after the lipid loading is required [5].


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)

The new microsyringe-based system for dispensing. A The new removable needle consisting of an Ito microsyringe needle (a) and a fitting ring (b). B A schematic diagram of the 96-hole polyester dual adhesive sheet (a) and the 96-hole spacer plate (b). Dimensions are given in millimeters. C The lipid-protein homogenization device with the monolithic stainless steel coupler
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: The new microsyringe-based system for dispensing. A The new removable needle consisting of an Ito microsyringe needle (a) and a fitting ring (b). B A schematic diagram of the 96-hole polyester dual adhesive sheet (a) and the 96-hole spacer plate (b). Dimensions are given in millimeters. C The lipid-protein homogenization device with the monolithic stainless steel coupler
Mentions: Homogenization of the lipid/water or lipid/protein buffer mixtures was performed at room temperatures (20–23 °C) by using a device consisting of a pair of Ito MS-GAN025 (250 μl) microsyringes with a homebuilt monolithic stainless steel coupler (Fig. 3C). The detailed structure and the homogenization procedures of this device were described in a previous paper [11], where the achievement of molecular homogeneity of lipid/water mixtures was confirmed by a small angle X-ray scattering measurements [10–12]. Though the homogenization procedures were essentially the same as those in the standard in meso protocol [5], there was one major difference in the lipid-loading step. The lipids used in this work are viscous waxy material over a temperature range from 0 to at lest ~80 °C [10, 12]. Thus, the lipids (usually 10–30 mg) were loaded into the open end of the microsyringe at room temperatures (without heating) by using a homebuilt stainless steal microspatula followed by an immediate homogenization with water or a protein buffer. In loading solid monoacylglycerols, e.g., monoolein, heating of the lipid at ~40 °C before or after the lipid loading is required [5].

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