DNA from phage lysates was extracted by phenol-chloroform and precipitated by 3 M sodium acetate and 100% ethanol as previously described [21 ] or following the manufacturer’s instructions of the Phage DNA Isolation Kit (Norgen Biotek). Briefly, 300 mL high titer lysate (1010 PFU/mL) was precipitated with 10% polyethylene glycol 8000 (Merck, Germany) and 1 M sodium chloride (Merck) followed by centrifugation for 30 min at 10,000 rpm (Sorvall RC 6 Plus with rotor F21S 8 × 50 y, Thermo ScientificTM, USA). The pellet was resuspended in 2–4 mL SM-buffer and treated with 10-fold reaction buffer (100 mM Tris-HCl (pH 7.5), 25 mM MgCl2, und 1 mM CaCl2, Thermo Fisher Scientific, USA), 0.2 mg/mL RNase A (Thermo Fisher Scientific, USA), and 0.002 U/µL DNase I (Thermo Fisher Scientific, USA) followed by incubation overnight at 37 °C and 300 rpm in a Thermomix (Eppendorf, Germany). DNA was isolated using phenol-chloroform extraction and precipitated by 3 M sodium acetate and 100% ethanol. After incubation for 15 min at −80 °C, DNA was pelleted by centrifugation and washed twice with 70% ethanol. The pellet was air-dried and solved in 50–200 µL TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8, Merck, Germany). The concentration was determined using the Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions.
Polyethylene glycol 8000
Manufactured by Merck Group
Sourced in United States, Germany, Macao
Polyethylene glycol 8000 is a high molecular weight polymer that is commonly used as a laboratory reagent. It is a white, water-soluble, and non-toxic solid. Polyethylene glycol 8000 has a range of applications in various scientific and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g, by Addgene (6 mentions)
Sodium chloride (nacl), by Merck Group (5 mentions)
Pspax2 packaging plasmid, by Addgene (4 mentions)
Opti mem medium, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Caffeine, by Merck Group (3 mentions)
Phenol, by Merck Group (3 mentions)
Sodium hydroxide (naoh), by Merck Group (3 mentions)
Polyethylenimine (pei), by Polysciences (3 mentions)
Acetic acid, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Methyl anthranilate, by Merck Group (2 mentions)
Trimethylamine n oxide tmao, by Merck Group (2 mentions)
Sorbitol, by Merck Group (2 mentions)
Benzyl alcohol, by Merck Group (2 mentions)
4 nitrophenyl d glucopyranoside, by Merck Group (2 mentions)
4 nitroanisole, by Thermo Fisher Scientific (2 mentions)
O phthaldialdehyde opa reagent complete, by Merck Group (2 mentions)
62 protocols using polyethylene glycol 8000
1
Phage DNA Extraction Protocol
2
Nanogel Design with PEG and LPEI
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The experimental procedures required the following polymers for the nanogel design: polyethylene glycol 8000 (Mw = 8 kDa, by Merck KGaA, Darmstadt, Germany) and linear polyethyleneimine 2500 (Mw= 2.5 kDa, by Polysciences Inc., Warrington, USA). All other chemicals were purchased from Merck (Merck KGaA, Darmstadt, Germany) and used as received, without any further purification.
Solvents were of analytical-grade purity. All the rhodamine derivatives were stored at 4°C.
Solvents were of analytical-grade purity. All the rhodamine derivatives were stored at 4°C.
3
HMGA2 Liquid-Liquid Phase Separation Assay
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The in vitro LLPS assay of full-length or mutant HMGA2 was performed for 10 min at 37 °C in a solution containing 12 mM Tris-HCl (pH 7.5−8.0), 0.2 mM dithiothreitol, 0.4 mM β-mercaptoethanol, and 5% glycerol and with various concentrations of HMGA2 and NaCl in the absence or presence of mononucleosomes (800 nM). Assay of PUB1IDR-HMGA2 hook1 del mutant was conducted in a solution containing 16 mM Tris-HCl (pH 7.5−8.0), 0.2 mM dithiothreitol, 300 mM NaCl, 1.2 mM β-mercaptoethanol, and 7% glycerol. Fluorescence and DIC images were acquired with a confocal microscope (Leica TCS-SP5 or Zeiss LSM 880) and were processed and analyzed with ImageJ (NIH). Turbidity was determined by measurement of OD400 with a Nano-drop ND-1000 instrument (Thermo Fisher). Polyethylene glycol 8000 (Sigma) was added to a final concentration of 10% for FRAP analysis.
4
Structural Analysis of ZIKV NS4B Protein
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Chemically synthesized ZIKV NS4B cytosolic region (NS4B-CR, 131-169 residues of NS4B-FL protein;
ZIKV strain UniProt ID: A0A024B7W1) (NH2-AARAAQKRTAAGIMKNPVVDGIVVTDIDMTIDPQVEKK-COOH) with 96% purity was purchased from Thermo scientific USA and dissolved in buffer consisting of 20 mM sodium phosphate buffer (pH 7.4). Chemicals including Sodium Dodecyl Sulfate, 2,2,2-Trifluoroethanol, Trimethylamine N-oxide, and Poly Ethylene Glycol 8000 were obtained from Sigma Aldrich, St. Louis, USA. Ficoll-70 was purchased from Cytiva, Marlborough, MA, USA. Liposomes were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, USA.
ZIKV strain UniProt ID: A0A024B7W1) (NH2-AARAAQKRTAAGIMKNPVVDGIVVTDIDMTIDPQVEKK-COOH) with 96% purity was purchased from Thermo scientific USA and dissolved in buffer consisting of 20 mM sodium phosphate buffer (pH 7.4). Chemicals including Sodium Dodecyl Sulfate, 2,2,2-Trifluoroethanol, Trimethylamine N-oxide, and Poly Ethylene Glycol 8000 were obtained from Sigma Aldrich, St. Louis, USA. Ficoll-70 was purchased from Cytiva, Marlborough, MA, USA. Liposomes were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, USA.
5
Solvatochromic Probes and Osmolytes
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Polyethylene glycol-8000 (Lot 091M01372V) with an average molecular weight (M w ) of 8,000 and polyethylene glycol-10000 (Lot 043K2522) with an average molecular weight (M w ) of 10,000 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvatochromic probes 4-nitrophenol (reagent grade, >98%) was purchased from Aldrich (Milwaukee, WI, USA) and 4-nitroanisole (>97%, GC) was received from Acros Organics. Reichardt's carboxylated betaine dye, 2,6-diphenyl-4-[2,6-diphenyl-4-(4-carboxyphenyl)-1-pyridino]phenolate, sodium salt was kindly provided by Professor C. Reichardt (Philipps University, Marburg, Germany).
Sorbitol and trimethylamine N-oxide (TMAO) were purchased from Sigma-Aldrich, and sucrose was received from USB (Cleveland, OH, USA). Benzyl alcohol, caffeine; coumarin, methyl anthranilate, 4-nitrophenyl-␣-d-glucopyranoside, phenol, 2-phenylethanol, vanillin, and o-phthaldialdehyde (OPA) reagent (complete) were purchased from Sigma-Aldrich. All compounds were of 98-99% purity and used as received without further purification. All salts and other chemicals used were of analytical-reagent grade.
Sorbitol and trimethylamine N-oxide (TMAO) were purchased from Sigma-Aldrich, and sucrose was received from USB (Cleveland, OH, USA). Benzyl alcohol, caffeine; coumarin, methyl anthranilate, 4-nitrophenyl-␣-d-glucopyranoside, phenol, 2-phenylethanol, vanillin, and o-phthaldialdehyde (OPA) reagent (complete) were purchased from Sigma-Aldrich. All compounds were of 98-99% purity and used as received without further purification. All salts and other chemicals used were of analytical-reagent grade.
6
RNA Extraction and Purification Protocol
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Tris hydrochloride (pH 9.0), sodium dodecyl sulfate (SDS), lithium chloride (LiCl), ethylenediaminetetraacetic acid (EDTA), phenol (pH 8.0), chloroform–isoamyl alcohol (24:1; v/v), phenol–chloroform–isoamyl alcohol (25:24:1; v/v/v), sodium acetate (3 M, pH 5.2), sodium chloride (NaCl), polyethylene glycol 8,000, and pepsin were obtained from Sigma‐Aldrich (St. Louis, MO). RNA oligos with 3′ end 2′‐O‐methylation were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
7
Preparation and Titration of HCV Virus Stock
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The HCV virus stock was obtained by concentrating the JFH-1 virion-containing supernatant through polyethylene glycol-8000 (Sigma-Aldrich) and its titer was determined via the immunofluorescence assay of HCV core-positive foci [29 (link)]. This prepared virus stock was later used for the infection of naïve Huh7.5 cells at the multiplicity of infection (MOI) of 0.01.
8
Generation of Dclk1-GFP Cell Lines
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As previously described46 (link), Dclk1 isoform long-α cDNA tagged with GFP and GFP cDNA was amplified and ligated into pCR8-GW-TopoD (Invitrogen), following the manufacturer’s protocol. Wild-type Dclk1-GFP and GFP cDNAs were then transferred to pLenti CMV PURO DEST empty (gift from Dr. Eric Campeau) using Clonase 2 (Invitrogen), following the manufacturer’s recommendations. The expression plasmids constructed above were co-transfected along with packaging plasmids pMD2.G (Addgene), pMDL/RRE g/p (Addgene), and pRSV-Rev (Addgene) into 293 T cells. DNA was transfected into cells using the PEI transfection method, with a PEI to DNA ratio of 1:1. Supernatants were harvested 48 and 72 h post-transfection and cleared through a 0.45-μm filter. These viral supernatants were concentrated using polyethylene glycol 8000 (Sigma-Aldrich). Cells were infected with concentrated virus and selected with puromycin (Sigma-Aldrich) to establish stable cell lines.
9
Solvatochromic Probes and Osmolytes
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Polyethylene glycol-8000 (Lot 091M01372 V) with an average molecular weight (M w ) of 8000 and polyethylene glycol-10000 (Lot 043K2522) with an average molecular weight (M w ) of 10000 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvatochromic probes 4-nitrophenol (reagent grade, >98%) was purchased from Aldrich (Milwaukee, WI, USA) and 4-nitroanisole (>97%, GC) was received from Acros Organics. Reichardt's carboxylated betaine dye, 2,6-diphenyl-4-[2,6diphenyl-4-(4-carboxyphenyl)-1-pyridino]phenolate, sodium salt was kindly provided by Professor C. Reichardt (Philipps University, Marburg, Germany).
Sorbitol, trimethylamine N-oxide (TMAO), and trehalose were purchased from Sigma-Aldrich, and sucrose was received from USB (Cleveland, OH, USA). 4-Aminophenol, benzyl alcohol, caffeine; coumarin, methylanthranilate, 4-nitrophenyl-␣-D-glucopyranoside, phenol, 2-phenylethanol, vanillin, and o-phthaldialdehyde (OPA) reagent (complete) were purchased from Sigma-Aldrich. All compounds were of 98-99% purity and used as received without further purification. All salts and other chemicals used were of analytical-reagent grade.
Sorbitol, trimethylamine N-oxide (TMAO), and trehalose were purchased from Sigma-Aldrich, and sucrose was received from USB (Cleveland, OH, USA). 4-Aminophenol, benzyl alcohol, caffeine; coumarin, methylanthranilate, 4-nitrophenyl-␣-D-glucopyranoside, phenol, 2-phenylethanol, vanillin, and o-phthaldialdehyde (OPA) reagent (complete) were purchased from Sigma-Aldrich. All compounds were of 98-99% purity and used as received without further purification. All salts and other chemicals used were of analytical-reagent grade.
10
SDS-PAGE Protein Analysis Protocol
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