Polyethylene glycol peg 8000

All reagents used were of analytical grade or better, and were used without further purification. Anhydrous Na₂HPO₄, NaH₂PO₄, CaCl₂ and NaCl were purchased from Dinâmica Química Contemporânea Ltd.a (Diadema/SP, Brazil). MgSO₄ was supplied by Sigma-Aldrich (St. Louis, MO, USA). TSA and TSB were purchased from Sigma-Aldrich Brazil (Cotia/SP, Brazil). Bacteriologic agar was from Gibco Diagnostics (Madison, WI, USA). Polyethylene glycol (PEG) 8000 was from Sigma-Aldrich (St. Louis, MO, USA). “Enterokit B” was acquired from PROBAC DO BRASIL Produtos Bacteriológicas Ltd.a. (São Paulo/SP, Brazil), and consisted of the following culture media: EPM, MILi and Simmons Citrate, in a box with 16 sets and a vial of Kovacs reagent. The Stericup™—GP sterilizing filtration systems (0.22 µm pore diameter polyethersulphate membrane) were acquired from Merck-Millipore (Darmstadt, Germany). Deodorized cocoa butter (lot. 04DS02) was purchased from BrazilCoa Com. de Prod. Ltd.a. (Indaiatuba/SP, Brazil). Ultrapure water was produced in a Master System All MS2000 (Gehaka, São Paulo/SP, Brazil) to a final resistivity of 0.1818 MΩ·m and conductivity of 5 µS·m⁻¹.

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids (IL, USA). 6-acetyl-2-dimethylaminonaphthalene (ACDAN) was purchased from Santa Cruz Biotechnology (USA), and 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) from Thermofisher Scientific (USA). ATTO 647N-DOPE was obtained from ATTO-TEC GmbH (Siegen, Germany). Polydimethylsiloxane (PDMS) and curing agent were obtained as part of the SYLGARD® 184 silicone elastomer kit from Dow Corning (Michigan, USA). Dextran from Leuconostoc spp (Mw 450–650 kg/mol), and poly(ethylene glycol) (PEG 8000, Mw 8 kg/mol) were purchased from Sigma-Aldrich. Chloroform obtained from Merck (Darmstadt, Germany) was of HPLC grade (99.8%). The lipid stocks were mixed as Chloroform solutions at 4 mM, containing 0.1 mol% ATTO 647N-DOPE or 0.5 mol% LAURDAN, and were stored until use at −20 °C. Fluorescein isothiocyanate isomer (FITC), sucrose, glucose, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH) and sodium chloride (NaCl) were obtained from Sigma-Aldrich (Missouri, USA). All aqueous solutions were prepared using ultrapure water from a SG water purification system (Ultrapure Integra UV plus, SG Wasseraufbereitung) with a resistivity of 18.2 MΩ cm.

Polyethylene glycol (PEG-8000, Lot# SLBW6815) with molecular weight (Mw) of 8000 Da, polypropylene glycol (PPG-400, Lot# BCBT9613) with Mw of 400 Da, and polyvinylpyrrolidone (PVP-40,000 Lot# WXBD4555V and Lot# WXBB3898V) with Mw of 40,000 Da were obtained from Sigma-Aldrich (St. Louis, MO, USA). Polyacrylamide (PAM-10,000, Lot# A804225) with Mw of 10,000 Da (50% wt. in water solution) was purchased from Polysciences, Inc. (Warrington, PA, USA). Ucon 50-HB-5100 (Ucon-3930, Lot# SJ1955S3D2), a random copolymer of 50% ethylene oxide and 50% propylene oxide, with Mw of 3930, was purchased from Dow-Chemical (Midland, MI, USA). Sodium sulfate, sodium phosphate monobasic, sodium phosphate dibasic, and KCl of analytical reagent grade were purchased from Fisher Scientific (Waltham, MA, USA) and used without further purification. Ultrapure water purified using a Millipore Milli-Q lab water system was used for preparation of all solutions.
Dinitrophenylated (DNP-) amino acids (DNP-alanine, DNP-norvaline, DNP-norleucine, and DNP-α-amino-N-octanoic acid) were purchased from Sigma-Aldrich. The sodium salts of the DNP-amino acids were prepared by titration.

ADP, PEP, oxalate (OX), F16BP, F26BP, lactate dehydrogenase (LDH; rabbit muscle), polyethylene glycol (PEG) 8000, antibiotics and buffers were obtained from Sigma-Aldrich. NADH and EDTA-free protease inhibitor mixture tablets were from Roche, glycerol was from BDH Prolabo, IPTG was from Melford and salts were from Fisher Scientific. Restriction enzymes, vector and E. coli competent cells were from Novagen.
N-terminal His₆-tagged TcPYK was prepared as described previously [28 (link)]. Briefly, the expression of His₆-tagged TcPYK was achieved by the T7lac promoter-driven system in E. coli BL21 (DE3) cells after adding IPTG to a final concentration of 1 mM. Pure protein was obtained by immobilized metal ion affinity chromatography (IMAC) followed by gel-filtration chromatography. N-terminally His₆-tagged human pyruvate kinases (M1PYK and M2PYK) were expressed in E. coli BL21(DE3) cells and purified using IMAC and gel-filtration chromatography as described previously [16 (link)].

Polyethylene glycol PEG-8000 with a number average molecular weight (Mn) of 8000 Da; polyethylene glycol PEG-10000 with Mn of 10,000 Da; polyethylene glycol 6000, Mn = 6000 Da; polyethylene glycol 4000, Mn = 4000 Da; polyethylene glycol 1000, Mn = 1000 Da, and polyethylene glycol 600, Mn = 600 Da were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Dextran-75 (Lot 119945) with an average molecular weight (Mw) of 75,000 Da by light scattering was purchased from USB Corporation (Cleveland, OH, USA). Ucon 50-HB-5100, Mw = 3930 Da was purchased from Dow-Chemical (Midland, MI, USA). Ficoll 70, Mw ~ 70,000 Da was purchased from GE Healthcare Biosciences AB (Sweden). All polymers were used without further purification.