After 24–26 hours of cultivation on LP medium, B. licheniformis cells were acidified with acetic acid to pH 5.0, centrifuged at 9000 ×g for 20 minutes at 4°C. The supernatant was diluted twice with sterile distilled water and was applied onto the DEAE-cellulose (Servacel, Germany) column (≈30 mL), equilibrated with 0.01 M Na-acetate buffer, pH 5.0. Then the solution was applied onto the phosphocellulose P-11 (Whatman, England) column (≈50 mL), equilibrated with the same buffer. After that the column was washed with 0.01 M Na-acetate buffer, pH 5.0, until optical density of eluate at 280 nm decreased below 0.05. Then the column was equilibrated with 0.01 M Na-phosphate buffer, pH 7.0. The elution was carried out with 0.2 M Na-phosphate buffer, pH 7.0. Fractions corresponding to RNase activity peak were combined and desalted using centrifugal filter units Ultracel-3K (Merck Millipore, USA). The additional purification was carried out using Biologic DuoFlow FPLC system (BioRad, USA) on the UNOS6 (BioRad, USA) column, equilibrated with 20 mM Na-acetate buffer, pH 5.0. Proteins were eluted using a linear gradient of 0-1 M NaCl.
Phosphocellulose p 11
Manufactured by Cytiva
Sourced in United Kingdom
Phosphocellulose P-11 is a cellulose-based ion exchange resin designed for the purification and separation of proteins, enzymes, and other biomolecules. It is a strong cation exchanger with a high capacity for binding positively charged molecules. The resin is suitable for use in a wide range of chromatographic techniques, including column chromatography and batch adsorption.
Automatically generated - may contain errors
Lab products found in correlation
Tryptone, by BD (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Yeast extract, by BD (2 mentions)
Sodium deoxycholate, by Merck Group (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Ssdna cellulose resin, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Hindiii, by New England Biolabs (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (2 mentions)
Biologic duoflow fplc system, by Bio-Rad (1 mentions)
Ultracel 3k, by Merck Group (1 mentions)
Unos6, by Bio-Rad (1 mentions)
Bio gel htp hydroxyapatite, by Bio-Rad (1 mentions)
Lactate dehydrogenase ldh, by Merck Group (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
Histrap ff, by GE Healthcare (1 mentions)
Deae sepharose fast flow, by GE Healthcare (1 mentions)
3 protocols using phosphocellulose p 11
1
Purification of RNase from B. licheniformis
2
Purification of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All chemicals were of
reagent grade, made
up in Nanopure water, and passed through 0.2 μm pore size filters.
Yeast extract and tryptone were from Becton Dickinson and Company
(MD, USA). NaCl, sucrose, Tris base, KCl, Na2HPO4, NaH2PO4, EDTA, acetic acid, methanol, and
nickel sulfate were from J.T. Baker (NJ, USA). Ampicillin was from
Fisher (NJ, USA). IPTG was from OmniPur (NJ, USA). Kanamycin, chloramphenicol,
lysozyme, and sodium deoxycholate were from Sigma (MO, USA). Benzonase
was from Novagen (NJ, USA). Imidazole was from EMD (NJ, USA). Coomassie
brilliant blue R-250 was from Bio-Rad Laboratories (CA, USA). Glucose
was from Mallinckrodt (KY, USA). Nonidet P40 substitute was from USB
(OH, USA). ATP and DEAE Sepharose Fast Flow (FF), Q-Sepharose, HisTrap
FF, 16/10 heparin FF, Mono Q, and Mono S 5/50 GL columns were from
GE Healthcare Life Sciences (NJ, USA). Phosphoenol pyruvate (PEP),
nicotinamide adenine dinucleotide (NADH), pyruvate kinase (PK), lactate
dehydrogenase (LDH), and ssDNA-cellulose resin were from Sigma. Phosphocellulose
(P11) was from Whatman. Bio-Gel HTP hydroxyapatite was from Bio-Rad.
Dithiothreitol (DTT) was from Acros Organics. BSA and HindIII were purchased from New England Biolabs. Wheat germ topoisomerase
I (WGT) was from Promega.
reagent grade, made
up in Nanopure water, and passed through 0.2 μm pore size filters.
Yeast extract and tryptone were from Becton Dickinson and Company
(MD, USA). NaCl, sucrose, Tris base, KCl, Na2HPO4, NaH2PO4, EDTA, acetic acid, methanol, and
nickel sulfate were from J.T. Baker (NJ, USA). Ampicillin was from
Fisher (NJ, USA). IPTG was from OmniPur (NJ, USA). Kanamycin, chloramphenicol,
lysozyme, and sodium deoxycholate were from Sigma (MO, USA). Benzonase
was from Novagen (NJ, USA). Imidazole was from EMD (NJ, USA). Coomassie
brilliant blue R-250 was from Bio-Rad Laboratories (CA, USA). Glucose
was from Mallinckrodt (KY, USA). Nonidet P40 substitute was from USB
(OH, USA). ATP and DEAE Sepharose Fast Flow (FF), Q-Sepharose, HisTrap
FF, 16/10 heparin FF, Mono Q, and Mono S 5/50 GL columns were from
GE Healthcare Life Sciences (NJ, USA). Phosphoenol pyruvate (PEP),
nicotinamide adenine dinucleotide (NADH), pyruvate kinase (PK), lactate
dehydrogenase (LDH), and ssDNA-cellulose resin were from Sigma. Phosphocellulose
(P11) was from Whatman. Bio-Gel HTP hydroxyapatite was from Bio-Rad.
Dithiothreitol (DTT) was from Acros Organics. BSA and HindIII were purchased from New England Biolabs. Wheat germ topoisomerase
I (WGT) was from Promega.
3
Purification of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were reagent grade, made up in Nanopure water and passed through 0.2 µm pore size filters. Yeast Extract and tryptone were from Becton Dickinson and Company (MD, USA).
NaCl, sucrose, tris-base, KCl, Na2HPO4, NaH2PO4, EDTA, acetic acid, methanol and nickel sulfate were from J.T. Baker (NJ, USA). Ampicillin was from Fisher (NJ, USA). IPTG was from OmniPur (NJ, USA). Kanamycin, chloramphenicol, lysozyme and sodium deoxycholate were from Sigma (MO, USA). Benzonase was from Novagen (NJ, USA). Imidazole was from EMD (NJ, USA). Coomassie brilliant blue R-250 was from Bio-Rad Laboratories (CA, USA). Glucose was from Mallinckrodt (KY, USA). Nonidet P40 substitute was from USB (OH, USA). ATP, DEAE Sepharose Fast Flow, Q-Sepharose, the HisTrap FF, the 16/10 heparin FF, Mono Q and the Mono S 5/50 GL columns were from GE Healthcare Life Sciences (NJ, USA). Phosphoenol pyruvate (PEP), nicotinamide adenine dinucleotide (NADH), pyruvate kinase (PK), lactate dehydrogenase (LDH) and the ssDNA-cellulose resin were from Sigma. Phosphocellulose (P11) was from Whatman. Bio-Gel ® HTP hydroxylapatite was from Bio-Rad. Dithiothreitol (DTT) was from Acros Organics. BSA and HindIII were purchased from New England Biolabs. Wheat Germ Topoisomerase I (WGT) was from Promega.
NaCl, sucrose, tris-base, KCl, Na2HPO4, NaH2PO4, EDTA, acetic acid, methanol and nickel sulfate were from J.T. Baker (NJ, USA). Ampicillin was from Fisher (NJ, USA). IPTG was from OmniPur (NJ, USA). Kanamycin, chloramphenicol, lysozyme and sodium deoxycholate were from Sigma (MO, USA). Benzonase was from Novagen (NJ, USA). Imidazole was from EMD (NJ, USA). Coomassie brilliant blue R-250 was from Bio-Rad Laboratories (CA, USA). Glucose was from Mallinckrodt (KY, USA). Nonidet P40 substitute was from USB (OH, USA). ATP, DEAE Sepharose Fast Flow, Q-Sepharose, the HisTrap FF, the 16/10 heparin FF, Mono Q and the Mono S 5/50 GL columns were from GE Healthcare Life Sciences (NJ, USA). Phosphoenol pyruvate (PEP), nicotinamide adenine dinucleotide (NADH), pyruvate kinase (PK), lactate dehydrogenase (LDH) and the ssDNA-cellulose resin were from Sigma. Phosphocellulose (P11) was from Whatman. Bio-Gel ® HTP hydroxylapatite was from Bio-Rad. Dithiothreitol (DTT) was from Acros Organics. BSA and HindIII were purchased from New England Biolabs. Wheat Germ Topoisomerase I (WGT) was from Promega.
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