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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

Repetitive measurements of the delivered volumes of a cubic phase, using a 25 μl microsyringe and a 21 gauge removable needle. The delivery conditions were: red circles 200 nl (240 pulses/trigger, L = 300 μm), black circles 100 nl (120 pulses/trigger, L = 200 μm), green circles 50 nl (60 pulses/trigger, L = 150 μm), and blue circles 25 nl (30 pulses/trigger, L = 125 μm)
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Fig5: Repetitive measurements of the delivered volumes of a cubic phase, using a 25 μl microsyringe and a 21 gauge removable needle. The delivery conditions were: red circles 200 nl (240 pulses/trigger, L = 300 μm), black circles 100 nl (120 pulses/trigger, L = 200 μm), green circles 50 nl (60 pulses/trigger, L = 150 μm), and blue circles 25 nl (30 pulses/trigger, L = 125 μm)

Mentions: Repetitive measurements were performed using 200 nl (red circles), 100 nl (black circles), 50 nl (green circles) and 25 nl (blue circles) as target dispensing volumes, as shown in Fig. 5. The average of the actually dispensed volumes, the standard deviation (σ) and the coefficient of variation (CV) for each target volume were 204 nl (σ = 2.6 nl, CV = 1.3 %), 97.4 (σ = 4.5 nl, CV = 4.6 %), 46.3 (σ = 6.0 nl, CV = 13 %), and 22.5 (σ = 5.0 nl, CV = 22 %), respectively. The results in Fig. 5 indicated that when a target volume was above 100 nl, the actual dispensed volume agreed reasonably well with the target volume (<5 %), whereas deviations from the desired values tended to occur as the target volume decreased, due in part to the complex rheological nature of the cubic phase. Thus, a correction factor may be applied when more accurate dispensing is required (see the next section). The smallest volume that could be dispensed with the present system was about 25 nl. However, the reproducibility was considerably lower than those of volumes ≥50 nl, and extreme perseverance was required to reach the reported reproducibility level. For these reasons, we considered ~50 nl to be the most reasonable lowest volume for the dispenser, to perform manual crystallization trials without great difficulty.Fig. 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)

Repetitive measurements of the delivered volumes of a cubic phase, using a 25 μl microsyringe and a 21 gauge removable needle. The delivery conditions were: red circles 200 nl (240 pulses/trigger, L = 300 μm), black circles 100 nl (120 pulses/trigger, L = 200 μm), green circles 50 nl (60 pulses/trigger, L = 150 μm), and blue circles 25 nl (30 pulses/trigger, L = 125 μm)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Repetitive measurements of the delivered volumes of a cubic phase, using a 25 μl microsyringe and a 21 gauge removable needle. The delivery conditions were: red circles 200 nl (240 pulses/trigger, L = 300 μm), black circles 100 nl (120 pulses/trigger, L = 200 μm), green circles 50 nl (60 pulses/trigger, L = 150 μm), and blue circles 25 nl (30 pulses/trigger, L = 125 μm)
Mentions: Repetitive measurements were performed using 200 nl (red circles), 100 nl (black circles), 50 nl (green circles) and 25 nl (blue circles) as target dispensing volumes, as shown in Fig. 5. The average of the actually dispensed volumes, the standard deviation (σ) and the coefficient of variation (CV) for each target volume were 204 nl (σ = 2.6 nl, CV = 1.3 %), 97.4 (σ = 4.5 nl, CV = 4.6 %), 46.3 (σ = 6.0 nl, CV = 13 %), and 22.5 (σ = 5.0 nl, CV = 22 %), respectively. The results in Fig. 5 indicated that when a target volume was above 100 nl, the actual dispensed volume agreed reasonably well with the target volume (<5 %), whereas deviations from the desired values tended to occur as the target volume decreased, due in part to the complex rheological nature of the cubic phase. Thus, a correction factor may be applied when more accurate dispensing is required (see the next section). The smallest volume that could be dispensed with the present system was about 25 nl. However, the reproducibility was considerably lower than those of volumes ≥50 nl, and extreme perseverance was required to reach the reported reproducibility level. For these reasons, we considered ~50 nl to be the most reasonable lowest volume for the dispenser, to perform manual crystallization trials without great difficulty.Fig. 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