3A6 Fab was crystallized in 28% PEG400, 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–sodium hydroxide (NaOH) pH 7.5 buffer, and 0.2 M calcium chloride (CaCl2) (Hampton Research, Viejo, California, USA) at 20°C. To form the 3A6 Fab–peptide complex, purified 3A6 Fab was concentrated to 5 mg/mL, combined with a five-fold excess of peptide, and incubated at 4°C for 18 h. The Fab–peptide complex was crystallized in 30% polyethylene glycol (PEG) 3000, 0.1 M tris(hydroxymethyl)aminomethane (Tris), pH 7.0, and 0.2 M sodium chloride (NaCl) (Hampton Research) at 20°C. Crystals were flash-cooled in liquid nitrogen, with 15% ethylene glycol (Hampton Research) as a cryoprotectant.
Hepes
Manufactured by Hampton Research
Sourced in United States
HEPES is a chemical buffer compound commonly used in cell culture and biochemical applications. It is a zwitterionic organic chemical compound that helps maintain a stable pH environment in aqueous solutions. HEPES is effective in maintaining a physiological pH range and is widely utilized in various research and laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using hepes
1
Crystallizing 3A6 Fab-Peptide Complex
2
Structural Analysis of CD99-10A1 Fab Complex
3
Crystallization of Zebrafish SPA17 NTD
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Extensive crystallization screening was conducted to obtain high-quality crystals. Purified D. rerio SPA17 NTD (1–75) protein was mixed with mother liquor in a 1:1 ratio using 150 nl of each component and spotted onto hanging drop seals on a Mosquito nanodrop robot. The crystallization trays were then placed at 4 °C. The crystals used for analysis grew in 0.1 M Hepes, pH 7.5, and 0.5 M magnesium formate dihydrate from the Index Screen by Hampton Research. Crystals appeared after 2 to 3 days and reached their final size in 1 to 2 weeks. Crystals were directly transferred to a cryo-protectant buffer containing 50% glycerol and 50% mother liquor and frozen in liquid nitrogen for synchrotron data collection.
4
A2AAR-STaR2-bRIL Protein Crystallization
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The receptor was
reconstituted into the lipidic cubic phase (LCP) by mixing two volumes
of purified receptor (30 mg mL–1) with three volumes
of molten monoolein/cholesterol (9:1 w/w using coupled gas-tight 100
μL syringes (Hamilton)).35 (link) LCP boli
(40 nL) were dispensed and overlaid with 800 nL of precipitant in
96-well LCP glass sandwich plates (Marienfeld) using the NT8-LCP system
(Formulatrix). Crystallization hits were obtained in 0.1 M HEPES (pH
5.3), 0.05 M sodium thiocyanate, 30% PEG400, and 2% (v/v) 2,2,2-trifluoroethanol
(Hampton Research). Crystals appeared within 6 days after setup and
reached their maximum size of 50 × 10 × 3 μm in the
case of the A2AAR-STaR2-bRIL construct
and within 2 days with the maximum size of 15 × 10 × 2 μm
for A2AAR-STaR2-S277-bRIL (Figure S1 ). Crystals were harvested directly
from LCP using 50 μm micromounts (MiTeGen) and flash-frozen
in liquid nitrogen.
reconstituted into the lipidic cubic phase (LCP) by mixing two volumes
of purified receptor (30 mg mL–1) with three volumes
of molten monoolein/cholesterol (9:1 w/w using coupled gas-tight 100
μL syringes (Hamilton)).35 (link) LCP boli
(40 nL) were dispensed and overlaid with 800 nL of precipitant in
96-well LCP glass sandwich plates (Marienfeld) using the NT8-LCP system
(Formulatrix). Crystallization hits were obtained in 0.1 M HEPES (pH
5.3), 0.05 M sodium thiocyanate, 30% PEG400, and 2% (v/v) 2,2,2-trifluoroethanol
(Hampton Research). Crystals appeared within 6 days after setup and
reached their maximum size of 50 × 10 × 3 μm in the
case of the A2AAR-STaR2-bRIL construct
and within 2 days with the maximum size of 15 × 10 × 2 μm
for A2AAR-STaR2-S277-bRIL (
from LCP using 50 μm micromounts (MiTeGen) and flash-frozen
in liquid nitrogen.
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