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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

Standard procedure for manual dispensing. A A 96-hole spacer plate (a) is superimposed on top of a 96-well glass crystallization plate. The front protecting sheet and the 96-hole double-stick sheet are denoted by b1 and b2, respectively. The center of the spacer plate hole and the center of the well are denoted by c1 and c2, respectively. B The removable needle is fully inserted into (a) the spacer hole. (b) The 96-hole double-stick sheet. (c) The glass plate. C A schematic drawing of the syringe tip (e) region, where the needle tip-to-glass plate distance L is fixed at a desired value by the relation , where , , and  are the thicknesses of the 96-hole double-stick sheet (b1 + b2), the 96-hole spacer plate (a), and the fitting ring (f), respectively.  is the syringe tip (e) length. The figures serve for illustrative purposes only, and are not drawn to scale
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Fig4: Standard procedure for manual dispensing. A A 96-hole spacer plate (a) is superimposed on top of a 96-well glass crystallization plate. The front protecting sheet and the 96-hole double-stick sheet are denoted by b1 and b2, respectively. The center of the spacer plate hole and the center of the well are denoted by c1 and c2, respectively. B The removable needle is fully inserted into (a) the spacer hole. (b) The 96-hole double-stick sheet. (c) The glass plate. C A schematic drawing of the syringe tip (e) region, where the needle tip-to-glass plate distance L is fixed at a desired value by the relation , where , , and are the thicknesses of the 96-hole double-stick sheet (b1 + b2), the 96-hole spacer plate (a), and the fitting ring (f), respectively. is the syringe tip (e) length. The figures serve for illustrative purposes only, and are not drawn to scale

Mentions: The homebuilt 96-hole spacer was made from a 2.95 mm thick acrylic plate, and contains 96 holes. The arrangement of the holes is identical to that of the 96-hole double-stick sheet, except the hole diameter is 5.9 mm, instead of 5 mm for the 96-hole double-stick sheet. Thus, the 96-hole spacer plate serves not only as a spacer to secure an optimum value of L, but also as a centering jig for dispensing mesophase boluses (see also Fig. 4).Fig. 4


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)

Standard procedure for manual dispensing. A A 96-hole spacer plate (a) is superimposed on top of a 96-well glass crystallization plate. The front protecting sheet and the 96-hole double-stick sheet are denoted by b1 and b2, respectively. The center of the spacer plate hole and the center of the well are denoted by c1 and c2, respectively. B The removable needle is fully inserted into (a) the spacer hole. (b) The 96-hole double-stick sheet. (c) The glass plate. C A schematic drawing of the syringe tip (e) region, where the needle tip-to-glass plate distance L is fixed at a desired value by the relation , where , , and  are the thicknesses of the 96-hole double-stick sheet (b1 + b2), the 96-hole spacer plate (a), and the fitting ring (f), respectively.  is the syringe tip (e) length. The figures serve for illustrative purposes only, and are not drawn to scale
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Standard procedure for manual dispensing. A A 96-hole spacer plate (a) is superimposed on top of a 96-well glass crystallization plate. The front protecting sheet and the 96-hole double-stick sheet are denoted by b1 and b2, respectively. The center of the spacer plate hole and the center of the well are denoted by c1 and c2, respectively. B The removable needle is fully inserted into (a) the spacer hole. (b) The 96-hole double-stick sheet. (c) The glass plate. C A schematic drawing of the syringe tip (e) region, where the needle tip-to-glass plate distance L is fixed at a desired value by the relation , where , , and are the thicknesses of the 96-hole double-stick sheet (b1 + b2), the 96-hole spacer plate (a), and the fitting ring (f), respectively. is the syringe tip (e) length. The figures serve for illustrative purposes only, and are not drawn to scale
Mentions: The homebuilt 96-hole spacer was made from a 2.95 mm thick acrylic plate, and contains 96 holes. The arrangement of the holes is identical to that of the 96-hole double-stick sheet, except the hole diameter is 5.9 mm, instead of 5 mm for the 96-hole double-stick sheet. Thus, the 96-hole spacer plate serves not only as a spacer to secure an optimum value of L, but also as a centering jig for dispensing mesophase boluses (see also Fig. 4).Fig. 4

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