Peg3350

Manufactured by Merck Group

Sourced in United States

PEG3350 is a polyethylene glycol compound used as a non-reactive, water-soluble polymer in various laboratory applications. It has a molecular weight of approximately 3,350 Daltons. PEG3350 is frequently employed as a component in buffer solutions, protein stabilizers, and other laboratory reagents.

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Lab products found in correlation

40 protocols using peg3350

Micro-crystal growth for LCLS injection

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Crystallization conditions were based on those previously described^{38 (link)}. Micro-crystals were grown using the seeded batch method. Two different conditions were used—10 mg ml⁻¹ protein concentration and 25% (wt/vol) PEG 3350 (Sigma-Aldrich, CAS 25322-68-3) and 15 mg ml⁻¹ and 30% (wt/vol) PEG 3350—and both sets of conditions included 0.1 M Tris (Sigma-Aldrich, CAS 77-86-1) pH 8.5, 0.2 M lithium sulfate (Sigma-Aldrich, CAS 10377-48-7) and 1.5% (vol/vol) seed. Seed stocks were prepared using either condition and subsequently filtered with 50- and 30-μm CellTrics filters (Sysmex Partec). Crystallization was performed in 2-ml round-bottom Eppendorf tubes, and the addition of reagents followed the order ‘buffer, precipitant, protein and seed’ in a final 1-ml volume followed by gentle mixing. Needle-shaped micro-crystals with dimensions of ~3 × 5 × 10–100 μm matured after 24 h at 20 °C. To reduce issues with blockages during crystal injection, especially with the Gas Dynamic Virtual Nozzle (GDNV) during LCLS^{40 (link)} injection, further size optimization was performed by breaking up the micro-crystals using glass beads (Supplementary Section 1 describes the procedure).

Hutchison C.D., Baxter J.M., Fitzpatrick A., Dorlhiac G., Fadini A., Perrett S., Maghlaoui K., Lefèvre S.B., Cordon-Preciado V., Ferreira J.L., Chukhutsina V.U., Garratt D., Barnard J., Galinis G., Glencross F., Morgan R.M., Stockton S., Taylor B., Yuan L., Romei M.G., Lin C.Y., Marangos J.P., Schmidt M., Chatrchyan V., Buckup T., Morozov D., Park J., Park S., Eom I., Kim M., Jang D., Choi H., Hyun H., Park G., Nango E., Tanaka R., Owada S., Tono K., DePonte D.P., Carbajo S., Seaberg M., Aquila A., Boutet S., Barty A., Iwata S., Boxer S.G., Groenhof G, & van Thor J.J. (2023). Optical control of ultrafast structural dynamics in a fluorescent protein. Nature Chemistry, 15(11), 1607-1615.

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Liquid-Liquid Phase Separation of SHP2

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For the LLPS assay, the purified SHP2^WT or SHP2^mut protein at 8 μM was mixed with a LLPS buffer containing 20 mM Tris pH 8.0, 12%(w/v) PEG3350 (Sigma) and incubated for 5 min at room temperature. Finally, 2 μL of each sample was pipetted onto a glass dish and imaged using a Leica microscope. Under the same conditions, 30 μL of each sample was added into 384-well white polystyrene plate with clear flat bottom, and the value of OD600 was measured by using a TECAN Infinite F PLEX Pro microplate reader (37 °C, 10 hrs).
For LLPS assays treated with allosteric inhibitor, 20/50μM SHP099 or ET070 was incubated with 8 μM SHP2^mut phase-separated droplets at 37 °C for 15 min, and further transferred to 384-well white polystyrene plate for OD600 kinetics study. The microplate reader reads every 5 minutes and shakes 10 seconds before each reading (37 °C, 10 hrs).
For SHP2^Y279C in the presence of 2P-IRS-1 peptide, 4 μM SHP2^Y279C protein was incubated with 1 μM peptide or DMSO at 37 °C for 15 min and further mixed with the LLPS buffer. Each sample was then subjected to OD600 kinetics study at 37 °C for 10 hrs.
Phase diagrams were generated by mixing SHP2 protein (varying from 0.125–8 μM, final) in phase separation buffer 20 mM Tris pH 8.0, 12%(w/v) PEG3350 (Sigma), sodium chloride (varying from 50–500 mM). Droplet turbidity OD600 was measured by microplate reader as described above.

Zhu G., Xie J., Kong W., Xie J., Li Y., Du L., Zheng Q., Sun L., Guan M., Li H., Zhu T., He H., Liu Z., Xia X., Kan C., Tao Y., Shen H.C., Li D., Wang S., Yu Y., Yu Z.H., Zhang Z.Y., Liu C, & Zhu J. (2020). Phase separation of disease-associated SHP2 mutants underlies MAPK hyperactivation. Cell, 183(2), 490-502.e18.

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Light Scattering Analysis of PEG-Protein Interactions

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The PEG3350 (M = 3,350 g/mol, purchased from Sigma) solution was prepared by dissolving solid PEG3350 in a proper amount of Milli-Q water. The purified NalD protein (1.0 mg/mL) and PEG3350 (400 mg/mL) solutions were clarified by 20-nm (Whatman, Anotop 10) and 0.45-µm hydrophilic PTFE (Millipore) filters, respectively, just before mixing them for LLS measurements. The solution mixtures of NalD and PEG (C_PEG = 0–10 mg/mL) were prepared by adding a proper amount of the clarified PEG solution and Milli-Q water into a fixed amount of the clarified NalD solution. Each solution mixture was measured by dynamic LLS (ALV/DLS/SLS-5022F) at a scattering angle of 90° at 20°C. Using only one angle is because the scattering intensity is nearly angular independent. The theory and other details of LLS instrumentation can be found elsewhere [14] .

Yu J., Chen W., Wu C, & Chen H. (2014). PEG-Protein Interaction Induced Contraction of NalD Chains. PLoS ONE, 9(5), e96616.

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Mosquito Repellent Formulation Development

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Cetyl alcohol, stearic acid, vanillin, Span 80, Tween 60, carboxymethyl cellulose (CMC), polyethylene glycol (PEG) 3350, benzalkonium chloride (BKC), and diethyl-m-toluamide (DEET) were purchased from Sigma-Aldrich, Saint Louis, USA. Dow corning 200 was purchased from Dow Corning, Michigan, USA. Meanwhile, jojoba oil, sweet almond oil, coconut oil, emulsifying wax, shea butter, and cocoa butter were purchased from BF1 Malaysia, Selangor, Malaysia. The mosquitoes as subjects in the repellent efficacy study were provided by the Institute for Medical Research of Malaysia (IMR). Susceptible strains and nulliparous three- to seven-day-old adults were used as test species.

Misni N., Mohamed Nor Z., Ahmad R., Ithnin N.R, & Zasmy Unyah N. (2021). Microencapsulation Preservation of the Stability and Efficacy of Citrus Grandis Oil-Based Repellent Formulation against Aedes aegypti during Storage. Molecules, 26(12), 3599.

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DNA Fragment Amplification and Purification

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Phusion DNA polymerase (Thermo Fisher, United States) was used to amply DNA fragments. PrimeSTAR GXL DNA Polymerase (Takara, Japan) was used to amply DNA fragments whose length are larger than 10 kb. Trans 5 K and 1 kb DNA markers (TransGen Biotech, Beijing) were used to measure the size of DNA fragments in agarose gel electrophoresis. Gel extraction kit (Omega, United States), Plasmid extraction mini Kit (Omega, United States), and TIANamp Bacteria DNA Kit (TianGen, China) were used to purify DNA fragments. All oligos were synthesized by Beijing Genomics Institute. Magnesium chloride, manganese chloride, PEG8000, PEG3350, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (US), and the rest reagents were purchased from Sangon Biotech (China).

Yang Y., Yu Q., Wang M., Zhao R., Liu H., Xun L, & Xia Y. (2022). Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies. Frontiers in Microbiology, 13, 838698.

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Labeling and Characterizing Protein Solubility

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Lyophilized protein was dissolved into dissolving buffer (2 M guanidine hydrochloride, 100 mM Tris–HCl pH 8.0 and 10 mM HEPES) to a final concentration of 4 mM. For fluorescence microscopic observation, protein was incubated with 10 µM ATTO488-maleimide (ATTO-TEC) at room temperature for 1 h and then with 5 mM dithiothreitol at room temperature for 1 h or at 4 °C overnight. The labelled protein solution was diluted in droplet buffer (50 mM HEPES, 100 mM NaCl and 15% (w/v) PEG3350 (Sigma-Aldrich), pH 7.4) at a 1:100 ratio, incubated at room temperature for 30 min and transferred to a 96-well clear-bottom plate (Greiner Bio-One) for microscopic observation (FV3000, Olympus). The final concentration of protein was 40 μM unless otherwise indicated. For protein with multiple repeats, the final protein concentration is indicated in the figure legend. For the turbidity assay, protein in dissolving buffer was sequentially diluted with the same buffer, and then mixed with droplet buffer at 1:50. The mixture was incubated at room temperature for 10 min and transferred to a microcuvette. The optical density at 600 nm (OD₆₀₀) was measured using a V-630 spectrophotometer (JASCO). The C_sat value was defined by the concentration at which the turbidity was at half-maximal value^{10 (link)}. The data obtained were fitted using the equation (Supplementary Note) in OriginPro (v.9.8).

Yamazaki H., Takagi M., Kosako H., Hirano T, & Yoshimura S.H. (2022). Cell cycle-specific phase separation regulated by protein charge blockiness. Nature Cell Biology, 24(5), 625-632.

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Genetic Manipulation of Yeast Strains

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S. cerevisiae strains K665 (Δvcx1Δpmc1) and OC06 (Δvcx1Δvnx; provided by O. Cagnac and K. Venema, Estación Experimental del Zaidín, Granada, Spain; Table S1) were transformed with empty piHGpd plasmid or piHGpd plasmids harboring CAX, CAXΔN, and CAX^E420A using the lithium acetate/polyethylene glycol method (Gietz and Schiestl, 2007 (link)) to generate K665 and OC06 strains each with ectopically integrated GAPDH_promoter-CAX, GAPDH_promoter-CAXΔN, and GAPDH_promoter-CAX^E420A. A 1-ml aliquot of yeast cells grown for 16 h in yeast extract–peptone–dextrose medium was pelleted, washed in a buffer containing 0.1 M lithium acetate, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, and incubated with 4 µg plasmid DNA, 0.1 mg denatured salmon sperm carrier DNA (Sigma-Aldrich), 33% (wt/vol) polyethylene glycol (PEG 3350; Sigma-Aldrich), and 0.1 M lithium acetate. After mixing, the cells were heat shocked by incubation at 42°C for 1 h while shaking and then transferred to 20°C, washed, and then resuspended in sterile water before selection growth. Selection was performed with synthetic defined medium minus histidine (SD-H; Takara Bio Inc.) by growth at 30°C on solid SD-H medium.

Melchionda M., Pittman J.K., Mayor R, & Patel S. (2016). Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo. The Journal of Cell Biology, 212(7), 803-813.

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Yeast Transformation with Linearized Plasmid

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All DNA samples and a linearized pRS415 were collected in a tube (0.5 – 4.0 μg for each DNA sample and 100 ng linearized pRS415 in 50 μl water), and mixed with the transformation mixture [50 μl yeast competent cell, 240 μl 50% PEG3350 (Sigma), 36 μl 1 M LiAc, 25 μl 2 mg/ml salmon sperm DNA (Sigma)]. The mixture was incubated at 30°C for 30 min, then at 42°C for 20 min or at 42°C for 45 min, centrifuged at 8,000 g for 15 sec, and suspended in 200 μl water. Transformants were selected on complete synthetic defined medium without leucine (SD-Leu) [0.67% YNB+Nitrogen (Sunrise Science Products), 0.069% CSM-Leu (Sunrise Science Products), 2% dextrose] agar plates at 30°C for 3 days.

Ando H., Lemire S., Pires D.P, & Lu T.K. (2015). Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell systems, 1(3), 187-196.

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Induction of Somatic Embryos in Presence of PEG

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After the multiplication phase, the cultures were transferred to maturation treatments. Three colonies, each with a fresh mass (FM) of 300 mg, were inoculated into Petri dishes (90 × 15 mm) containing 20 mL of MS culture medium supplemented with myo-inositol (Merck KGaA, Darmstadt, Germany) (0.005%), Phytagel (2.0 g/L), sucrose (3%) and PEG 3350 (Sigma-Aldrich) at concentrations of 0, 3, 6 and 9%, hereafter referred to as the control, PEG3, PEG6 and PEG9 treatments, respectively. The pH of the culture medium was adjusted to 5.8 before the Phytagel was added. The culture medium was sterilized by autoclaving at 121°C for 15 min. The cultures were incubated in a growth chamber at 25 ± 1°C in the dark for the first 7 days, after which they were subjected to a 16 h light (60 μmol/m² s¹) photoperiod. Four repetitions were performed, with a total of 12 colonies per treatment. After 42 days of cultivation, the number of cotyledonary somatic embryos (mature somatic embryos) per colony was evaluated. Samples of 300 mg (FM) were dried in an oven at 70°C for 48 h for dry matter (DM) determination. Samples of 300 mg FM were also stored at -20°C for proteomic analysis.

Vale E.D., Heringer A.S., Barroso T., Ferreira A.T., da Costa M.N., Perales J.E., Santa-Catarina C, & Silveira V. (2014). Comparative proteomic analysis of somatic embryo maturation in Carica papaya L.. Proteome Science, 12, 37.

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RmsI Enzyme Co-Crystallization Protocol

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The truncation mutant RmsI (G12-P258) was concentrated to 0.42 mM (13 mg/ml) in a solution containing 50 mM Tris-HCl pH8.5 and 80 mM NaCl. AdoMet (Sigma, USA, 180 mM stock solution) and RmsI were mixed at a molar ratio of 5:1 and incubated on ice for 6 h before performing co-crystallization experiments. The crystallization screen was performed by mixing 1μl RsmI-AdoMet mixture and 1μl well buffer in the 48-well XtalQuest crystallization plate (MiTeGen, USA). Crystals were grown at 291K using the sitting-drop vapor diffusion method. The final crystallization condition is 0.2 M DL-Malic acid (pH 7.0), 20% PEG3350 (Sigma, USA).

Zhao M., Zhang H., Liu G., Wang L., Wang J., Gao Z., Dong Y., Zhang L, & Gong Y. (2016). Structural Insights into the Methylation of C1402 in 16S rRNA by Methyltransferase RsmI. PLoS ONE, 11(10), e0163816.

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