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Gryphon lcp

Manufactured by Art Robbins Instruments
Sourced in United States

The Gryphon LCP is a laboratory instrument designed for precision liquid chromatography. It provides accurate and consistent sample handling and analysis capabilities to support scientific research and testing applications.

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7 protocols using gryphon lcp

1

Muscarinic Receptor Crystallization by LCP

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Purified M1-T4L•tiotropium and M4-mT4L•tiotropium were crystallized using lipid cubic phase technology. Each receptor was reconstituted by mixing the protein solution into 10:1 (w/w) monoolein:cholesterol (Sigma) in 1:1.5 parts w/w protein:lipid ratio using the two-syringe method24 (link). For the M1 receptor, samples of 50 nL (20–40 nL for M4) were spotted onto 96-well glass plates and overlaid with 800 nL (600 nL for M4) of precipitant solution for each well using a Gryphon LCP (Art Robbins Instruments). Glass plates were then sealed using a glass cover film and incubated at 20 °C. Initial crystals for the M1 receptor formed after 24 hours in conditions containing 33% PEG 300, 100 mM sodium acetate, and 100 mM Bis-Tris Propane (pH 8.0). For the M4 receptor, initial crystals formed after 24 hours in conditions containing 25–40% PEG 300, 50–100 mM EDTA (pH 8.0), and 100 mM MES (pH 5.5–6.5). M1 and M4 crystals were harvested using mesh grid loops (MiTeGen) and stored in liquid nitrogen before use.
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2

Crystallization of Scribble PDZ1:TMIGD1 Complex

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Scrib/PDZ1:TMIGD1 PBM peptide complexes were prepared for crystallisation trials as described88 (link). The protein:peptide complexes were resuspended in buffer A at a molar ratio of 1:4. Using an in-house Gryphon LCP liquid dispenser (Art Robbins Instruments), initial sparse matrix crystallisation trials were performed in 96-well sitting drop trays (Swissci AG, Neuheim, Switzerland) with 0.2 μl of protein sample and 0.2 μl reservoir per drop. Subsequent crystal optimisations were carried out in 24-well Limbro plates (Hampton Research). All crystallisation trials were performed at 20 °C. Human Scribble PDZ1:Human TMIGD1 PBM crystals were obtained at 8 mg/ml in 24% w/v PEG 1500, flashed cool in 20% ethylene glycol.
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3

Crystallization of YeeE Protein

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Crystals of YeeE appeared in lipidic cubic phase (LCP). A solution of 300 mM sodium thiosulfate was added to the purified protein solution (about 20 mg/mL YeeE) to the final concentration of 75 mM. The solution was mixed with monoolein (Nu-Chek Prep) at a 2:3 ratio (w/w) using the twin-syringe mixing method (25 (link)). Each 50 nl of the mixture was spotted on crystallization plates and covered by 3 μl of a reservoir solution using a crystallization robot (Gryphon LCP, Art Robbins Instruments). Crystals of native YeeE and SeMet-labeled YeeE were grown up to about 80 μm (long axis) and 30 μm (short axis) at 20°C in a reservoir solution containing 24 to 26% Pentaerythritol-propoxylate (5/4 PO/OH), 100 mM 2-morpholinoethanesulfonic acid (MES)–NaOH (pH 6.5 to 7.0), and 100 to 150 mM NaCl for 5 days. The crystals were harvested using Crystal Mounts and Loops (MiTegen), directly flash cooled in liquid nitrogen, and stored in liquid nitrogen until the x-ray diffraction experiments.
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4

Structural Determination of Rh-PDE Crystals

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The purified protein was concentrated to 10 mg /mL and mixed with monoolein (Nu-Chek Prep) in a 2:3 protein to lipid ratio (w/w)45 (link). Aliquots of the protein-LCP mixture were dispensed onto 96-well glass plates and overlaid with the precipitant solution, using a Gryphon LCP (Art Robbins Instruments, LLC). Crystals of Rh-PDE (TMD) were obtained in 29–35% (w/v) PEG400, 100 mM Na-citrate, pH 5.0, 100 mM KSCN and 10 mM ZnCl2, and crystals of Rh-PDE (TMD–Linker) were obtained in 25% (w/v) PEG500DME, 100 mM Na-citrate, pH 5.0, 100 mM NaK-tartrate. All crystals were incubated at 20 °C for 1 week in the dark. The crystals were harvested using micromounts (MiTeGen) and were flash-cooled in liquid nitrogen without any additional cryoprotectants.
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5

Protein Crystallization Optimization Protocol

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The protein was concentrated to ∼10 mg ml−1 for crystallographic experiments. Initially, crystallization screenings were performed using a Gryphon LCP crystallization robot (Art Robbins Instruments, USA) in 96-well plates (SWISSCI, Switzerland) using the sitting-drop vapour-diffusion technique (1:1 or 1:2 precipitant:protein ratio) at 20°C. Several commercial screens were used in the experiment.
After one week, crystal growth was monitored under the microscope and plausible conditions were further tested at 18°C in 15-well plates (EasyXtal, USA) using the hanging-drop vapour-diffusion technique (1:1, 1:1.5 or 1:2 precipitant:protein ratios with 500 µl precipitant solution in the reservoir). Different PEG concentrations were tested in this screening. After one week, the best crystallization conditions were chosen. Crystals were found in two solutions, the first composed of 0.02 M KH2PO4, 0.1 M bis-Tris propane pH 6.5, 22% PEG 3350 and the second composed of 0.2 M ammonium acetate, 0.1 M bis-Tris pH 5.5, 20% PEG 3350. The crystals obtained in these conditions were cooled in liquid nitrogen in cryosolutions corresponding to the crystallization solutions supplemented with 20% glycerol.
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6

Protein Crystal Growth via Sitting Drop

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Aliquots of the protein were dispensed onto 96-well sitting drop plates (Swissci) and overlaid with the precipitant solution, using a Gryphon LCP (Art Robbins Instruments, LLC). Crystals were obtained in 29% (v/v) PEG400, 50 mM calcium acetate, pH 5.0, and 200 mM NaCl. All crystals were incubated at 4 °C for 1 week in the dark. Crystals were harvested using micromounts (MiTeGen), supplemented with 15% ethylene glycol as an additional cryoprotectant, and flash-cooled in liquid nitrogen.
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7

Protein Crystallization Optimization Protocol

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The protein was concentrated to ∼10 mg ml−1 for crystallographic experiments. Initially, crystallization screenings were performed using a Gryphon LCP crystallization robot (Art Robbins Instruments, USA) in 96-well plates (SWISSCI, Switzerland) using the sitting-drop vapour-diffusion technique (1:1 or 1:2 precipitant:protein ratio) at 20°C. Several commercial screens were used in the experiment.
After one week, crystal growth was monitored under the microscope and plausible conditions were further tested at 18°C in 15-well plates (EasyXtal, USA) using the hanging-drop vapour-diffusion technique (1:1, 1:1.5 or 1:2 precipitant:protein ratios with 500 µl precipitant solution in the reservoir). Different PEG concentrations were tested in this screening. After one week, the best crystallization conditions were chosen. Crystals were found in two solutions, the first composed of 0.02 M KH2PO4, 0.1 M bis-Tris propane pH 6.5, 22% PEG 3350 and the second composed of 0.2 M ammonium acetate, 0.1 M bis-Tris pH 5.5, 20% PEG 3350. The crystals obtained in these conditions were cooled in liquid nitrogen in cryosolutions corresponding to the crystallization solutions supplemented with 20% glycerol.
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