Avicel (microcrystalline cellulose Sigma Aldrich PH101) was swollen with phosphoric acid to generate phosphoric acid swollen cellulose (PASC) as previously described47 with a few modifications: 4 grams of Avicel were suspended in 100 mL of phosphoric acid (85% w/v) at 40 °C and magnetically stirred for 1 hour. The mixture was then poured into 1900 mL of water and kept at 40 °C with further stirring for 1 hour. The suspension was left stationary to allow the fibers to sediment before decanting the supernatant. The suspension was washed four times with 2 L H2O (MilliQ-quality), 2 times with 2 L of a 1% NaHCO3 solution to reduce acidity, and then three additional times with 2 L H2O (MilliQ-quality) and stored at 4 °C until further use. The final cellulose content of the PASC suspension, was determined by enzymatic hydrolysis (24 hours, 50 °C) with a high dosage of Celluclast 1.5L cellulolytic enzymes and Novozym 188, followed by the determination of released glucose, leading to an estimated cellulose content of 1.5% w/v. The average DP of the PASC was determined by measuring the total number of reducing ends48 and compare this to the total amount of monomeric glucose, giving a degree of polymerization of 52.
Avicel
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, China, United Kingdom, Macao, Israel
Avicel is a microcrystalline cellulose product manufactured by Merck Group. It is a fine, white, odorless, and tasteless powder. Avicel serves as a binder, disintegrant, and diluent in the formulation of pharmaceutical, nutraceutical, and dietary supplement products.
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Lab products found in correlation
Carboxymethylcellulose, by Merck Group (8 mentions)
Xylan, by Merck Group (5 mentions)
T4 dna ligase, by New England Biolabs (4 mentions)
Lichenan, by Megazyme (3 mentions)
α cellulose, by Merck Group (3 mentions)
Cellobiose, by Merck Group (3 mentions)
Laminarin, by Merck Group (3 mentions)
Carboxymethylcellulose cmc, by Merck Group (3 mentions)
Restriction endonuclease, by Takara Bio (3 mentions)
Dna purification kit, by Takara Bio (3 mentions)
La taq dna polymerase, by Takara Bio (3 mentions)
Lichenin, by Megazyme (3 mentions)
Xyloglucan, by Megazyme (3 mentions)
Birchwood xylan, by Merck Group (3 mentions)
P nitrophenyl β d glucopyranoside, by Merck Group (3 mentions)
Potassium dihydrogen phosphate, by Merck Group (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Manganese sulphate, by Merck Group (2 mentions)
Gel extraction kit, by Thermo Fisher Scientific (2 mentions)
Glycine, by Merck Group (2 mentions)
118 protocols using avicel
1
Preparation of Phosphoric Acid Swollen Cellulose
2
Locust Bean Gum and Enzymatic Hydrolysis
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Locust bean gum, galactomannan extracted from the seeds of a locust bean tree with mannose: galactose ratio of approximately 4:1, was purchased from Adamas (Pennsylvania Avenue, Washington, DC). Xylan, avicel, and carboxy methyl cellulose (CMC) were purchased from Merck (Singapore), and lichenan was purchased from Megazyme (Ireland). All other chemicals used in this study were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (China).
The Tma MSB8 (ATCC 43589) strain was acquired from the American Culture Collection (Manassas, Virginia), and grown in the Thermotoga basal medium (TMB) supplemented with 0.5% glucose (Jiang et al., 2006 (link)). Host E. coli JM109 was purchased from Promega Corporation (Madison, WI, United States), whereas the pHsh vector was provided by Shine E Biotech (Nanjing, China).
The Tma MSB8 (ATCC 43589) strain was acquired from the American Culture Collection (Manassas, Virginia), and grown in the Thermotoga basal medium (TMB) supplemented with 0.5% glucose (Jiang et al., 2006 (link)). Host E. coli JM109 was purchased from Promega Corporation (Madison, WI, United States), whereas the pHsh vector was provided by Shine E Biotech (Nanjing, China).
3
Cell Culture and Infection Assays
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BHK-21, HeLa (CCL-2; ATCC), HEK293T (CRL-3216; ATCC), Vero E6 cells (a kind gift from C Drosten, Charité Berlin), and TZM-bl cells (obtained from the NIH AIDS Reagent Program) were cultured in DMEM with 10% FBS, 100 µg/ml streptomycin, and 2 mM L-glutamine. For an agarose-overlay, pre-warmed DMEM with 2% FBS mixed with liquid SeaPlaque Agarose (Lonza) to a final concentration of 0.8% agarose was added to cells 2 h postinfection. For plaque assays, Vero E6 cells were overlayed with 2.5% Avicel (Merck) after 1 h of virus inoculation. HEK293T cells expressing vector or IFITM-HA proteins were generated via retroviral transduction and subsequent puromycin selection. Interferon stimulation was performed using indicated concentrations of Roferon (Interferon-α2a; Roche).
4
Controlled Release Buspirone Formulation
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Buspirone powder was purchased from Tehran Darou Pharmaceuticals (Tehran, Iran). Cellulose acetate with 40% acetyl groups (Fluka, Swizerland) was used as a semipermeable membrane. Hydroxylpropyl methylcellulose (HPMC) (K4M) (Celeron, England), and Polyvinylpirrolidine (PVP K30) (Mowiol, Germany) were used as water swellable and gelling agents. NaCl (Merck Co., Dermastat, Germany) was applied as osmotic active agent. Other chemicals such as Avicel, sodium lauryl sulfate (SLS), magnesium stearate (MgS), PEG-400, potassium dihydrogen phosphate (KH2PO4), sodium hydroxide (NaOH), acetone and ethanol were obtained from Merck Co., Dermastat, Germany.
5
Enzymatic Characterization of TtMan5A
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The substrate specificity of the recombinant TtMan5A was examined using polysaccharides as the substrates, by measuring the liberated reducing sugars as described above, with the exception that the reactions were performed at 50 °C. Carob galactomannan was purchased from Megazyme International (Wicklow, Ireland) and Avicel from Merck KGaA (Darmstadt, Germany). Other substrates used in this study were purchased from Megazyme International, Merck KGaA, and Sigma Chemical Co. as previously described.17) (link)25) (link)
6
Enzymatic Hydrolysis of Cellulose and Steviol Glycosides
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Cellobiohydrolase I (CBH1) and endoglucanase 7 (EG7) were purchased from Megazyme (Bray, Co. Wicklow, Ireland). Avicel was purchased from Merck KGaA, Darmstadt, Germany. Stevioside (>85%) and Rebaudioside A (>98%) were purchased from TCI America (Boston, MA, United States). All other chemicals were purchased from Merck KGaA, Darmstadt, Germany, and they were of the highest purity available.
7
Synthesis of para-Methylbenzoyl Cellulose
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para-Methylbenzoyl cellulose (PMBC) was prepared by reaction of microcrystalline cellulose (Avicel, Merck, Darmstadt, Germany) with an excess of 4-methylbenzoyl chloride in a mixture of pyridine and triethylamine in the presence of a catalytic quantity of 4-(dimethylamino)pyridine, as previously described [8 (link)].
8
Cellulomonas uda Growth Conditions
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The strains used were C. uda (DSM 20108) purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and Cellulomonas sp. ATCC 21399 (DSM 20108) obtained from the American Type Culture Collection (Manassas, Virginia, USA). These two strains are identical and should both be referred to as C. uda.
The basal growth medium (referred to as BM) contained (per litre): NaCl, 1.5 g; (NH3)2SO4, 6.2 g; (Na)2HPO4, 9.1 g; KH2PO4, 0.9 g; EDTA, 50 mg; MgSO4·7H2O, 0.2 g; ZnSO4·7H2O, 8 mg; FeSO4·7H2O, 20 mg; MnSO4·H2O, 15 mg; CaCl2·2H2O, 26 mg; MOPS, 41.8 g; and yeast extract, 300 mg. Prior to autoclaving (121 °C for 20 min), the pH was adjusted to 7.4 with 5 M NaOH. Biotin (1 mg l−1) and thiamine (1 mg l−1) were aseptically added to the autoclaved medium from filter-sterilized stock solutions. Cellulosic growth substrates, α-cellulose (Sigma, C-8002) and Avicel (Merck, Microcrystalline, 1.02331.0500), were added prior to autoclaving, whereas the soluble growth substrates, glucose and cellobiose, were added aseptically to the autoclaved medium through a 0.22-µm filter.
The basal growth medium (referred to as BM) contained (per litre): NaCl, 1.5 g; (NH3)2SO4, 6.2 g; (Na)2HPO4, 9.1 g; KH2PO4, 0.9 g; EDTA, 50 mg; MgSO4·7H2O, 0.2 g; ZnSO4·7H2O, 8 mg; FeSO4·7H2O, 20 mg; MnSO4·H2O, 15 mg; CaCl2·2H2O, 26 mg; MOPS, 41.8 g; and yeast extract, 300 mg. Prior to autoclaving (121 °C for 20 min), the pH was adjusted to 7.4 with 5 M NaOH. Biotin (1 mg l−1) and thiamine (1 mg l−1) were aseptically added to the autoclaved medium from filter-sterilized stock solutions. Cellulosic growth substrates, α-cellulose (Sigma, C-8002) and Avicel (Merck, Microcrystalline, 1.02331.0500), were added prior to autoclaving, whereas the soluble growth substrates, glucose and cellobiose, were added aseptically to the autoclaved medium through a 0.22-µm filter.
9
Evaluating Mannanase Substrate Specificity
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The substrate specificity of crude mannanase was determined by incubating the crude enzymes with 1% (w/v) of various substrates under our identified optimal conditions. The substrates include LBG (Sigma-Aldrich, St. Louis, MO, USA), guar gum, (Sigma-Aldrich, St. Louis, MO, USA), carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA), xylan from larchwood (Sigma-Aldrich, St. Louis, MO, USA), xylan from oat spelts (Sigma-Aldrich, St. Louis, MO., USA), xylan from birchwood (Sigma-Aldrich, St. Louis, MO, USA), Avicel (Merck, Darmstadt, Germany), and potato starch (Sigma-Aldrich, St. Louis, MO, USA). Then, the reducing sugar was determined using DNS method as described above. The highest mannanase activity was used as a baseline for relative activity comparison.
The effect of different chemicals on enzyme activity was performed in the presence of the tested chemicals in the enzyme activity assays under optimal conditions for each crude extract. The enzyme activity without any additional chemical was used as a baseline for relative activity comparison.
The effect of different chemicals on enzyme activity was performed in the presence of the tested chemicals in the enzyme activity assays under optimal conditions for each crude extract. The enzyme activity without any additional chemical was used as a baseline for relative activity comparison.
10
Clitocybe cinerea RNA Extraction and Sequencing
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C. cinerea was grown in Kremer and Wood medium [15] (link) containing 2% Avicel (Merck, Whitehouse Station, NJ) for 12 d, and then total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with an oligo(dT) primer (Takara Bio, Shiga, Japan). PCR using KOD plus version 2 DNA polymerase (Toyobo, Osaka, Japan) with the synthesized first-strand cDNA as a template was primed with oligonucleotides flanking the predicted translational start and stop codons (20 nucleotides upstream,5′-TCGGGACCGACCACGAACG-3′ ; 63 nucleotides downstream, 5′-CGATTCTGTCTTGAAGCCCGACT-3′ ). The PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI) followed by sequencing with a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The nucleotide sequence reported in this paper has been deposited in the DDBJ database under accession number AB901366.
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C. cinerea was grown in Kremer and Wood medium [15] (link) containing 2% Avicel (Merck, Whitehouse Station, NJ) for 12 d, and then total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with an oligo(dT) primer (Takara Bio, Shiga, Japan). PCR using KOD plus version 2 DNA polymerase (Toyobo, Osaka, Japan) with the synthesized first-strand cDNA as a template was primed with oligonucleotides flanking the predicted translational start and stop codons (20 nucleotides upstream,
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