Crystallization was carried out in 96-well sitting-drop vapor diffusion plates in MRC format (Molecular Dimensions) at 18 °C and set up using a mosquito HTS robot (TTP Labtech). Typical drop ratios of 200 nL + 200 nL and 400 nL + 400 nL (protein solution + reservoir solution) were used for coarse screening and fine screening, respectively. For co-crystallization experiments, methylated UCHL1 (meUCHL1, 17 mg/mL) was preincubated with 1.2 equivalents of GK13S. Covalent adduct formation was confirmed by intact protein mass spectrometry. After buffer exchange into buffer G (20 mM Tris pH 8.0, 100 mM NaCl), meUCHL1~GK13S was concentrated at 30 mg/mL and crystallized in 2.3 M ammonium sulfate, 110 mM K3PO4, and 90 mM K2HPO4 as hexagonal prisms (120 × 45 × 45 μm3). Cryoprotection was achieved by placing the crystal for a few seconds into 3 M sodium malonate pH 7.0 with 1 mM GK13S, followed by immediate vitrification in liquid nitrogen.
Mosquito hts robot
Manufactured by SPT Labtech
The Mosquito HTS robot is a high-precision liquid handling system designed for automated pipetting and dispensing of small volumes. It is capable of accurately transferring nanoliter to microliter volumes across a range of microplates and tubes. The Mosquito HTS robot is intended for use in high-throughput screening and other life science applications requiring precise liquid handling.
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Lab products found in correlation
2 protocols using mosquito hts robot
1
Covalent UCHL1-GK13S Adduct Crystallization
2
Crystallization of ParD2:ParE2-Nb Complexes
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ParD2:ParE2-His in 20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM tris(2carboxyethyl)phosphine was concentrated to 12 mg ml -1 . To make super-complexes with nanobodies, concentrated ParD2:ParE2 was mixed with a molar excess of nanobody and incubated for 30 min at RT. The mix was subsequently injected onto a Superdex 200 16/60 equilibrated in SEC buffer. Fractions corresponding to ParD2:ParE2:Nb super-complexes were pooled and concentrated. SDS-PAGE was performed to check for the formation of supercomplexes.
Crystallization conditions were screened using a Mosquito HTS robot from TTP Labtech (http://ttplabtech.com/) using 0.1 µl protein solution and 0.1 µl of reservoir solution equilibrated against 100 µl of reservoir solution in sitting drop configuration and later on incubated at 292 K. Crystals grew after two years in 0.1 M KCl, 0.1 M Na-HEPES pH 7, 15 % PEG 5000 MME. For data collection, crystals were flash-cooled in liquid nitrogen. Data were collected on PROXIMA-2A at the SOLEIL synchrotron facility, Gif-sur-Yvette, France. All data were indexed, integrated and scaled with XDS (Kabsch, 2010) . Data quality and twinning were analyzed with phenix.xtriage (Liebschner et al., 2019) and POINTLESS (Evans, 2006) .
Analysis of solvent contents was performed using the CCP4 program MATTHEWS_COEF (Kantardjieff & Rupp, 2003) .
Crystallization conditions were screened using a Mosquito HTS robot from TTP Labtech (http://ttplabtech.com/) using 0.1 µl protein solution and 0.1 µl of reservoir solution equilibrated against 100 µl of reservoir solution in sitting drop configuration and later on incubated at 292 K. Crystals grew after two years in 0.1 M KCl, 0.1 M Na-HEPES pH 7, 15 % PEG 5000 MME. For data collection, crystals were flash-cooled in liquid nitrogen. Data were collected on PROXIMA-2A at the SOLEIL synchrotron facility, Gif-sur-Yvette, France. All data were indexed, integrated and scaled with XDS (Kabsch, 2010) . Data quality and twinning were analyzed with phenix.xtriage (Liebschner et al., 2019) and POINTLESS (Evans, 2006) .
Analysis of solvent contents was performed using the CCP4 program MATTHEWS_COEF (Kantardjieff & Rupp, 2003) .
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