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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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A Repetitive measurements of the delivered volumes of a cubic phase with the conditions of P = 35 pulses/trigger and L = 135 μm. The average actually dispensed volumes, the standard deviation and the coefficient of variation were 50.4, 4.8 nl, and 9.5 %, respectively. B Typical images of the delivered cubic phase boluses of 10 consecutive 50 nl dispensed boluses (bar 500 μm). C Repetitive measurements of the deviation, r, of the delivered bolus center with respect to the well center. D The definition of r for a delivered bolus center (black cross) with respect to the well center (red cross). The well diameter is 5 mm. The figure is not drawn to scale
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Fig6: A Repetitive measurements of the delivered volumes of a cubic phase with the conditions of P = 35 pulses/trigger and L = 135 μm. The average actually dispensed volumes, the standard deviation and the coefficient of variation were 50.4, 4.8 nl, and 9.5 %, respectively. B Typical images of the delivered cubic phase boluses of 10 consecutive 50 nl dispensed boluses (bar 500 μm). C Repetitive measurements of the deviation, r, of the delivered bolus center with respect to the well center. D The definition of r for a delivered bolus center (black cross) with respect to the well center (red cross). The well diameter is 5 mm. The figure is not drawn to scale

Mentions: The actual volumes of the delivered cubic phase boluses were quantified by processing microscopic images of delivered boluses, which were captured by a BX51 microscope equipped with a C-4040 digital camera (Olympus Co., Tokyo, Japan) [7]. As seen from typical bolus images in Fig. 6B, the bottom faces of delivered boluses were approximately circular in shape after being sandwiched between the two glass plates. We therefore assumed that the delivered boluses are circular truncated cones, so that the bolus volume, V, could be estimated from the equation, , where , , and d denote the upper and lower bottom face areas of the delivered bolus and the well thickness, respectively. The S values of each bolus image were quantified by the polygonal area analysis of Image-J (Bioarts Co., Fukuoka, Japan). Each image was calibrated with a NOB1 (pitch 0.01 mm) objective micrometer (Nikon, Co., Tokyo, Japan) to express the S value in the SI unit. The d value of each well was measured by a VK-8500 confocal microscopy (Kyence, Osaka, Japan) and was found to be 135 ± 2 μm.


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)

A Repetitive measurements of the delivered volumes of a cubic phase with the conditions of P = 35 pulses/trigger and L = 135 μm. The average actually dispensed volumes, the standard deviation and the coefficient of variation were 50.4, 4.8 nl, and 9.5 %, respectively. B Typical images of the delivered cubic phase boluses of 10 consecutive 50 nl dispensed boluses (bar 500 μm). C Repetitive measurements of the deviation, r, of the delivered bolus center with respect to the well center. D The definition of r for a delivered bolus center (black cross) with respect to the well center (red cross). The well diameter is 5 mm. The figure is not drawn to scale
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125823&req=5

Fig6: A Repetitive measurements of the delivered volumes of a cubic phase with the conditions of P = 35 pulses/trigger and L = 135 μm. The average actually dispensed volumes, the standard deviation and the coefficient of variation were 50.4, 4.8 nl, and 9.5 %, respectively. B Typical images of the delivered cubic phase boluses of 10 consecutive 50 nl dispensed boluses (bar 500 μm). C Repetitive measurements of the deviation, r, of the delivered bolus center with respect to the well center. D The definition of r for a delivered bolus center (black cross) with respect to the well center (red cross). The well diameter is 5 mm. The figure is not drawn to scale
Mentions: The actual volumes of the delivered cubic phase boluses were quantified by processing microscopic images of delivered boluses, which were captured by a BX51 microscope equipped with a C-4040 digital camera (Olympus Co., Tokyo, Japan) [7]. As seen from typical bolus images in Fig. 6B, the bottom faces of delivered boluses were approximately circular in shape after being sandwiched between the two glass plates. We therefore assumed that the delivered boluses are circular truncated cones, so that the bolus volume, V, could be estimated from the equation, , where , , and d denote the upper and lower bottom face areas of the delivered bolus and the well thickness, respectively. The S values of each bolus image were quantified by the polygonal area analysis of Image-J (Bioarts Co., Fukuoka, Japan). Each image was calibrated with a NOB1 (pitch 0.01 mm) objective micrometer (Nikon, Co., Tokyo, Japan) to express the S value in the SI unit. The d value of each well was measured by a VK-8500 confocal microscopy (Kyence, Osaka, Japan) and was found to be 135 ± 2 μm.

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