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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

Schematic diagram of the stepping motor-based dispenser
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Fig2: Schematic diagram of the stepping motor-based dispenser

Mentions: Figure 2 shows a schematic diagram of the stepping motor-based dispenser. As an actuator for dispensing, we employed a KSS model MB0601 5-phase stepping motor (TS3664N17E4) (KSS Co., Ltd. Tokyo, Japan), which moves by 2 μm per pulse (a). The actuator was housed in a 35 mm diameter, cylindrical plastic case. The microsyringe (b), in which the desired mesophase was loaded, was fixed at three positions, c1 (the barrel head), c2 and c3 by two magnetic clamps. The plunger head (d) was fixed at a slider (e) by a setscrew (f). The trigger switch (g) is for the actuator. The cubic phase loaded within the microsyringe is expelled onto the glass plate by a motor-driven plunger motion through a removable needle (h). The dispenser can accept any microsyringe type, but we here used Ito gas tight microsyringes, an MS-GFN25 (25 μl), an MS-GFN50 (50 μl), and an MS-GFN100 (100 μl), for which a full stroke is 60 mm (=6.0 × 104 μm). By using the values of 2 μm per pulse and 6.0 × 104 μm for full stroke length, the number of pulses per trigger, P, to deliver a desired volume, v(nl), can be obtained by the following relation,1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P = \frac{{ 3\times \text{10}^{\text{4}} v}}{{V_{S} }},$$\end{document}where Vs (nl) denotes the syringe volume employed for dispensing. For example, with a 100 μl (Vs = 105 nl) syringe, the P values to dispense 25, 50, and 100 nl of cubic phase are 8, 15, and 30 pulses/trigger, respectively. With a 50 μl (Vs = 5 × 104 nl) syringe, the corresponding P values are 15, 30 and 60 pulses/trigger, respectively.Fig. 2


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)

Schematic diagram of the stepping motor-based dispenser
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Schematic diagram of the stepping motor-based dispenser
Mentions: Figure 2 shows a schematic diagram of the stepping motor-based dispenser. As an actuator for dispensing, we employed a KSS model MB0601 5-phase stepping motor (TS3664N17E4) (KSS Co., Ltd. Tokyo, Japan), which moves by 2 μm per pulse (a). The actuator was housed in a 35 mm diameter, cylindrical plastic case. The microsyringe (b), in which the desired mesophase was loaded, was fixed at three positions, c1 (the barrel head), c2 and c3 by two magnetic clamps. The plunger head (d) was fixed at a slider (e) by a setscrew (f). The trigger switch (g) is for the actuator. The cubic phase loaded within the microsyringe is expelled onto the glass plate by a motor-driven plunger motion through a removable needle (h). The dispenser can accept any microsyringe type, but we here used Ito gas tight microsyringes, an MS-GFN25 (25 μl), an MS-GFN50 (50 μl), and an MS-GFN100 (100 μl), for which a full stroke is 60 mm (=6.0 × 104 μm). By using the values of 2 μm per pulse and 6.0 × 104 μm for full stroke length, the number of pulses per trigger, P, to deliver a desired volume, v(nl), can be obtained by the following relation,1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P = \frac{{ 3\times \text{10}^{\text{4}} v}}{{V_{S} }},$$\end{document}where Vs (nl) denotes the syringe volume employed for dispensing. For example, with a 100 μl (Vs = 105 nl) syringe, the P values to dispense 25, 50, and 100 nl of cubic phase are 8, 15, and 30 pulses/trigger, respectively. With a 50 μl (Vs = 5 × 104 nl) syringe, the corresponding P values are 15, 30 and 60 pulses/trigger, respectively.Fig. 2

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