Synthesis of pullulan-histone antibody nanoconjugates was performed as described in our recent publication using H4 IgG (Rekha et al. 2013 ). The process involved three steps, namely, (1) activation of pullulan: typically pullulan (Sigma Aldrich™, USA) was dissolved in 20 mM borax buffer, mixed with Traut’s reagent (2-Iminothiolane, Sigma Aldrich™, USA) under continuous stirring (final pH 7.0) and dialysed against 0.1 M sodium phosphate (pH 7.4) containing 0.15 M NaCl and 1 mM EDTA; (2) activation of H4 IgG: H4 IgG (200 μg/mL) was mixed with 3-maleimido benzoyl NHS (Sigma Aldrich™, USA) and kept for half an hour at 25°C for the reaction to be completed; (3) conjugation of activated pullulan with activated H4 IgG: activated H4 IgG was conjugated by vigorous stirring with activated pullulan to form monodispersed nanoconjugates.
Pullulan
Manufactured by Merck Group
Sourced in United States, Germany
Pullulan is a natural polysaccharide that is produced by the fungus Aureobasidium pullulans. It has a linear structure consisting of maltotriose units joined by α-1,6 glycosidic linkages. Pullulan is a water-soluble, odorless, and tasteless material that has a wide range of applications in the pharmaceutical, food, and cosmetic industries.
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Lab products found in correlation
Amylopectin, by Merck Group (4 mentions)
Laminarin, by Merck Group (3 mentions)
Glycogen, by Merck Group (3 mentions)
Amylose from potato, by Merck Group (3 mentions)
Starch, by Merck Group (3 mentions)
Ultimate iso 3100sd pump, by Thermo Fisher Scientific (3 mentions)
Wps 3000 sampler, by Thermo Fisher Scientific (3 mentions)
Alginate, by Merck Group (2 mentions)
Dextrin, by Merck Group (2 mentions)
Maltose, by Merck Group (2 mentions)
α cyclodextrin, by Merck Group (2 mentions)
Potato starch, by Merck Group (2 mentions)
Pullulan, by Megazyme (2 mentions)
Soluble starch, by Merck Group (2 mentions)
Amylopectin from maize, by Merck Group (2 mentions)
Amylopectin from potato, by Merck Group (2 mentions)
Potato amylopectin, by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Maltose, by Fujifilm (1 mentions)
44 protocols using pullulan
1
Pullulan-Histone Antibody Nanoconjugate Synthesis
2
Pullulan Cryogels for Antibiotic Release
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Pullulan (Alfa Aesar, Great Britain, Heysham, UK) and divinyl sulfone (DVS, 98%, Merck, Japan) were used in the synthesis of Pullulan cryogels. Ciprofloxacin (Recordati, Pharmaceuticals, Istanbul, Turkey) was used as a model drug in the drug release studies. Gram-negative bacterium Escherichia coli (ATCC 8739) and Gram-positive bacterium Staphylococcus aureus (ATCC 6538) were obtained from KWIK-STIK, Microbioloics. Nutrient agar (NA, Condolab, Madrid, Spain) as a solid growth medium and nutrient broth (NB, Merck, Darmstadt, Germany) as a liquid medium were used as received. As the cell culture, the L929 fibroblast cell line (Mouse C3/An connective tissue) was obtained from the SAP Institute, Ankara, Turkey. Dulbecco’s modified Eagle’s medium (DMEM) (4500 mg/L glucose, 3.7 g/L sodium pyruvate, and 0.5 g/mL L-glutamine) as the cell culture medium, fetal bovine serum (FBS, heat inactivated), and trypsin-EDTA (0.25%) were purchased from PanBiontech (A GmbH, Aidenbach, Germany). For the calorimetric cell viability analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from neoFroxx (A GmbH, Hesse, Germany) and trypan blue (0.5% solution) was obtained from Biological Industries. Dimethyl sulfoxide (DMSO, 99.9%, Carlo-Erba, Val-de-Reuil, France) was used in the cytotoxicity studies.
3
Analyzing Pullulan Hydrolysis by Recombinant PulB
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The hydrolysis products produced by the recombinant PulB protein were analyzed using thin-layer chromatography (TLC). The recombinant PulB (0.4 μM) was incubated with 0.25% pullulan (Sigma, St. Louis, MO), maltotriose (Nakarai tesque, Kyoto, Japan), maltose (Wako, Osaka, Japan), and glucose (Wako) in 50 mM sodium phosphate buffer (pH 6.2) at 37°C for 24 h. Five microliter aliquots of each reaction mixture were spotted onto a Silica gel 60 TLC plate with concentrating zone (Millipore, Darmstadt, Germany). The plate was developed with a 2-propanol/acetic acid/water (4:1:1) solvent system. The plates were dried and sprayed with orcinol-sulphuric acid reagent. The plate was heated at 120°C for 10 min.
4
Enzyme-Based Substrate Cleavage Assay
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PrimeSTAR high-fidelity DNA polymerase, T4 DNA ligase, and restriction enzymes were purchased from Takara (Otsu, Japan). ClonExpress II One Step Cloning Kit was provided by Vazyme (Nanjing, China). The substrate 4-nitrophenyl palmitate, substrate 4-nitrophenyl-α-L-arabinofuranoside, substrate pullulan and thrombin were supplied by Sigma-Aldrich (Milwaukee, USA). Other molecular biology reagents and chemicals (analytical grade) were obtained commercially.
5
Cloning and Characterization of Cohnella sp. A01 Carbohydrate-Active Enzymes
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DNA extraction kits were purchased from Bioneer Company (Seoul, Korea). The restriction enzymes, NdeI, NotI, and plasmid pTZ57R/T were prepared from Fermentas (Glen Burnie, MD, USA). Plasmid extraction kit was procured from Roche Company (Switzerland). Bacterial strains including E. coli DH5α and E. coli BL21 (DE3), the expression vector pET-26b (+), and Ni-NTA resin were purchased from Invitrogen (Carlsbad, USA) and pustulan from Invivogen Company (San Diego, CA, USA). Laminarin, pullulan, starch, cellulose, and sucrose were obtained from Sigma (St. Louis, USA). Other chemicals utilized in this study were prepared from Merck (Darmstadt, Germany). Cohnella sp. A01 was obtained from shrimp pond waste water at Choebdeh (Abadan, Iran; accession No. JN208862.1)[21 (link)]. VMD 1.9 (University of Illinois, Urbana-Champaign, USA), Gene Runner 4.0 (Lynnon Biosoft, Canada), and Graphpad Prism 6 (San Diego, CA, USA) software were used to analyze protein structure and DNA sequence as well as to draw Michaelis-Menten curve and to calculate Km and Vmax. The phylogenetic tree was established using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT)[22 (link)].
6
Pullulan-based Piroxicam Delivery System
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Piroxicam(4-Hydroxy-2-methyl-3-(pyrid-2-yl-carbamoyl)-2H-1,2-benzothiazine 1,1-dioxide, meets USP testing specifications), pullulan, PUL (from Aureobasidium pullulans, Mw 360–480 kDa), dextran sulfate sodium salt (Mw 7–20 kDa, sulfur content (S/C-analysis) 17.0–19.0%), (3-Acrylamidopropyl)trimethylammonium chloride solution–APTMAC (75 wt% in H2O), benzoyl peroxide (BPO) and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich. Spectroscopic grade methanol and oleic acid (p.a.) were purchased from POCH, Gliwice, Poland.
7
Polysaccharide-based Growth Substrates
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Starch was purchased from Merck (Germany). Yeast extract, peptone, gelatin, mono- and disaccharides, barley glucan, birchwood and beechwood xylan, carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC) Avicel, inulin, cellobiose, dextrin, dextran, pullulan, laminarin, lichenan, pectin, and alginate were purchased from Sigma Aldrich (Taufkirchen, Germany), or kindly provided by Dr. R. Wohlgemuth. Agarose (agarose MP) was purchased from Boehringer (Mannheim, Germany) and chitin (crab chitin) from Bioprogress (Russia). Chitin and chitosan were kindly provided by Dr. S. Lopatin from the Centre of Bioengineering, Research Center of Biotechnology, RAS, Moscow, Russia. Amorphous chitin (AMCH) and amorphous cellulose (AMC) for growth experiments and native activity measurements were prepared according to Sorokin et al. (2015) (link). Other polysaccharides, such as glucomannan, galactomannan, arabinoxylan, and curdlan, were purchased from Megazyme (Ireland). Bamboo leaves collected near the sampling site were dried at room temperature and used as the growth substrate.
8
Fenugreek Seed Extraction and Analysis
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Fenugreek (Trigonella foenum-graecum) seeds were purchased from local market.
DEAE-cellulose, glycogen, amylopectin, maltose, pullulan, α-cyclodextrin, β-cyclodextrin, trypsin profile 1GD kit were purchased from Sigma Chemical Co., St. Loius; Mo, U.S.A.
Molecular markers for FPLC were from Pharmacia, Sweden.
All solutions were prepared in Milli Q (Millipore, Bedford, MA, U.S.A.) water.
All the chemicals for buffer were of analytical or electrophoresis grade from Merck Eurolab GmbH Darmstadt, Germany.
DEAE-cellulose, glycogen, amylopectin, maltose, pullulan, α-cyclodextrin, β-cyclodextrin, trypsin profile 1GD kit were purchased from Sigma Chemical Co., St. Loius; Mo, U.S.A.
Molecular markers for FPLC were from Pharmacia, Sweden.
All solutions were prepared in Milli Q (Millipore, Bedford, MA, U.S.A.) water.
All the chemicals for buffer were of analytical or electrophoresis grade from Merck Eurolab GmbH Darmstadt, Germany.
9
Starch Utilization Optimization Protocol
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D-(+)-maltose monohydrate (Sigma-Aldrich) was used to stimulate growth from overnight stocks and as a model substrate for upregulating starch utilization. The maltodextrins used in this study were mixtures of maltodextrins with dextrose equivalent 16.5–19.5 (MD1) (Sigma-Aldrich) and maltodextrins with dextrose equivalent 4.0–7.0 (MD2) (Sigma-Aldrich). The soluble substrates potato starch, pullulan, amylopectin, amylose, dextran, and glycogen were purchased from Sigma-Aldrich (St. Louis, MO, USA), as well as the insoluble cornstarch. The cyclodextrin series (α-, β-, and γ-) were purchased from the American Maize Products Company (Stamford, CT, USA). Native potato starch was purchased from Bob’s Red Mill (Milwaukie, OR, USA). HIGH-MAIZE 260 cornstarch and VERSAFIBE 2470 were provided free of charge by Ingredion, Inc. (Westchester, IL, USA).
10
Monoclonal Antibody Characterization Protocol
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Hydrochloric acid (HCl, ACS reagent, 37%, Sigma Aldrich, St. Louis, MO, USA), sodium hydroxide (NaOH, >97%, Sigma Aldrich), sodium chloride (NaCl, >99.5%, Sigma Aldrich), acetic acid (AA, 100%, Roth, Karlsruhe, Germany), potassium dihydrogen phosphate (>99%, Roth), dipotassium hydrogen phosphate (>99%, Roth), pullulan (standard set, Mw 320–740,000 g/mol, Sigma Aldrich) were used in the following experiments. Ultrapure (UP) water was produced by an arium®pro ultra-pure water system (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The monoclonal antibodies (mAb1 (MW: 145.5 kDa; isoelectric point (pI): 8.7) and mAb2 (MW: 144.2 kDa; pI: 8.3)) were kindly donated by Sartorius Stedim Biotech.
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