Ad6-Luc was PEGylated as in ref. 35 (link). Following CsCl centrifugation, the preparation was desalted on EconoPac 10-DG columns (BioRad, Hercules, CA) into 0.5 M sucrose in PBS (136 mmol/l NaCl, 2.6 mmol/l KCl, 1.7 mmol/l KH2PO4, 10 mmol/l K2HPO4). 5 or 20 kDa succinimide-activated PEG (NOF America, White Plains, NY) was reacted with Ad6 at 10 mg/ml PEG for 1 hour at room temperature with rotation. Unreacted PEG was removed on a Sephadex G50 (GE Healthcare, Piscataway, NJ) size exclusion column. Mock-treated Ad5 was treated with no addition of PEG and treated in parallel.
Econopac 10dg columns
Manufactured by Bio-Rad
Sourced in United States
EconoPac® 10DG columns are size exclusion chromatography columns designed for the rapid desalting and buffer exchange of protein samples. They are pre-packed with Bio-Gel® P-6 resin and available in a convenient, ready-to-use format.
Automatically generated - may contain errors
Lab products found in correlation
Chlorotoxin, by Alomone (3 mentions)
Sephadex g 50, by GE Healthcare (2 mentions)
Methyl peg8 nh2, by Thermo Fisher Scientific (2 mentions)
Xl blue, by Agilent Technologies (1 mentions)
Ni nta agarose column, by Qiagen (1 mentions)
Pqe30, by Qiagen (1 mentions)
Optilab t rex refractometer, by Wyatt Technology (1 mentions)
Dna sequencing, by Eurofins (1 mentions)
Kta purifier, by GE Healthcare (1 mentions)
11 protocols using econopac 10dg columns
1
PEGylation and Purification of Adenovirus 6
2
Radiolabeling DOTA-Conjugated Antibody with Lutetium-177
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The chelator p-SCN-Bn-DOTA (DOTA) was dissolved in 0.005 M HCl, added to the antibody in a 6∶1 ratio and pH-adjusted to approximately 8.5 using a carbonate buffer. After 45 minutes of incubation at 37°C the reaction was stopped by the addition of 50 µl of 0.2 M glycine solution per mg of Ab. To remove free DOTA the conjugated antibody was washed using AMICON-30 centrifuge tubes (Millipore, Cork, Ireland) 4–5 times with NaCl 0.9%. Before labeling with 177Lu the pH was adjusted to 5.3±0.3 using 0.25 M ammonium acetate buffer. Between 120 and 220 MBq of 177Lu (Perkin Elmer, Boston, Ma, USA) were added to 1 mg of DOTA-Ab, and incubated for 45 minutes at 37°C. The radiochemical purity (RCP) of the conjugate was evaluated using instant thin layer chromatography. If RCP was below 95% the conjugate was purified using Econo-Pac 10 DG columns (Bio-rad Laboratories, California, USA)
3
Acid Extraction of C. jejuni Antigens
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An acid extract (AE) of C. jejuni strains was prepared as described
elsewhere with minor modification [14 (link)]. In brief,
bacterial cells grown on Brucella agar plates were harvested in 10 mM phosphate-buffered
saline (PBS, pH 7.2) and centrifuged at 8,000 × g at 4°C for 10 min. The
pellet was washed three times with PBS. Then, 3 ml of 100 mM glycine-hydrogen chloride
(Nacalai Tesque, Kyoto, Japan) buffer (pH 2.2) was added. The suspensions were stirred
with a magnetic stirrer for 30 min, and centrifuged at 18,000 × g at 4°C
for 10 min. Then, the supernatant was neutralized using Econo-Pac® 10DG Columns
(Bio-Rad Laboratories, Tokyo, Japan). The antigen concentration was determined using a
Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories) in accordance with the
manufacturer’s instructions. Each AE from 4 strains was adjusted to the same concentration
and 10 µg/ml AE was prepared as the ELISA antigen mixture. The pooled AEs were stored at
−80°C until further work.
elsewhere with minor modification [14 (link)]. In brief,
bacterial cells grown on Brucella agar plates were harvested in 10 mM phosphate-buffered
saline (PBS, pH 7.2) and centrifuged at 8,000 × g at 4°C for 10 min. The
pellet was washed three times with PBS. Then, 3 ml of 100 mM glycine-hydrogen chloride
(Nacalai Tesque, Kyoto, Japan) buffer (pH 2.2) was added. The suspensions were stirred
with a magnetic stirrer for 30 min, and centrifuged at 18,000 × g at 4°C
for 10 min. Then, the supernatant was neutralized using Econo-Pac® 10DG Columns
(Bio-Rad Laboratories, Tokyo, Japan). The antigen concentration was determined using a
Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories) in accordance with the
manufacturer’s instructions. Each AE from 4 strains was adjusted to the same concentration
and 10 µg/ml AE was prepared as the ELISA antigen mixture. The pooled AEs were stored at
−80°C until further work.
4
Bioconjugation of PBdots with Biomolecules
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Example 24
For biomolecular conjugation, tumor-specific peptide ligand chlorotoxin (CTX) was purchased from Alomone Labs, Ltd. (Jerusalem, Israel). Streptavidin was purchased from Invitrogen (Eugene, Oreg., USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and amine terminated polyethylene glycol (Methyl-PEG8-NH2) was purchased from Thermo Fisher Scientific (Rockford, Ill., U.S.A.).
Bioconjugation was performed by utilizing the EDC-catalyzed reaction between carboxyl PBdots and the respective amine-containing biomolecules (Methyl-PEG8, —NH2, chlorotoxin, and Streptavidin). In a typical conjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mL of carboxyl PBdot solution (50 μg/mL in MilliQ water), resulting in a PBdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL of amine-containing molecules (1 mg/mL) was added to the solution and mixed well on a vortex. Last, 60 μL of freshly-prepared EDC solution (5 mg/mL in MilliQ water) was added to the solution, and the above mixture was magnetically stirred for 4 hours at room temperature. Finally, the resulting PBdot-CTX and PBdot-PEG conjugates were separated from free molecules by Bio-Rad EconoPac® 10DG columns (Hercules, Calif., USA). PBdot-Streptavidin bioconjugates were separated by gel filtration using Sephacryl HR-300 gel media.
5
Bioconjugation of PBdot Nanoparticles
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Example 24
For biomolecular conjugation, tumor-specific peptide ligand chlorotoxin (CTX) was purchased from Alomone Labs, Ltd. (Jerusalem, Israel). Streptavidin was purchased from Invitrogen (Eugene, OR, USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and amine terminated polyethylene glycol (Methyl-PEG8-NH2) was purchased from Thermo Fisher Scientific (Rockford, IL, U.S.A).
Bioconjugation was performed by utilizing the EDC-catalyzed reaction between carboxyl PBdots and the respective amine-containing biomolecules (Methyl-PEG8,-NH2, chlorotoxin, and Streptavidin). In a typical conjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mL of carboxyl PBdot solution (50 μg/mL in MilliQ water), resulting in a PBdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL of amine-containing molecules (1 mg/mL) was added to the solution and mixed well on a vortex. Last, 60 μL of freshly-prepared EDC solution (5 mg/mL in MilliQ water) was added to the solution, and the above mixture was magnetically stirred for 4 hours at room temperature. Finally, the resulting PBdot-CTX and PBdot-PEG conjugates were separated from free molecules by Bio-Rad EconoPac® 10DG columns (Hercules, CA, USA). PBdot-Streptavidin bioconjugates were separated by gel filtration using Sephacryl HR-300 gel media.
6
Bioconjugation of PBdots with Tumor-Targeting Ligands
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Example 24
For biomolecular conjugation, tumor-specific peptide ligand chlorotoxin (CTX) was purchased from Alomone Labs, Ltd. (Jerusalem, Israel). Streptavidin was purchased from Invitrogen (Eugene, Oreg., USA). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and amine terminated polyethylene glycol(Methyl-PEG8-NH2) was purchased from Thermo Fisher Scientific (Rockford, Ill., U.S.A).
Bioconjugation was performed by utilizing the EDC-catalyzed reaction between carboxyl PBdots and the respective amine-containing biomolecules (Methyl-PEG8-NH2, chlorotoxin, and Streptavidin). In a typical conjugation reaction, 60 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 60 μL of concentrated HEPES buffer (1 M) were added to 3 mL of carboxyl PBdot solution (50 μg/mL in MilliQ water), resulting in a PBdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 30 μL of amine-containing molecules (1 mg/mL) was added to the solution and mixed well on a vortex. Last, 60 μL of freshly-prepared EDC solution (5 mg/mL in MilliQ water) was added to the solution, and the above mixture was magnetically stirred for 4 hours at room temperature. Finally, the resulting PBdot-CTX and PBdot-PEG conjugates were separated from free molecules by Bio-Rad EconoPac® 10DG columns (Hercules, Calif., USA). PBdot-Streptavidin bioconjugates were separated by gel filtration using Sephacryl HR-300 gel media.
7
Recombinant Human MANF Protein Production
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Recombinant human MANF protein expression and purification were conducted as described previously (Wen et al., 2006 (link)). The open reading frame of mature human MANF cDNA was cloned into an expression vector pQE30 (QIAGEN), fused to a 6xHis tag at the amino terminus to generate plasmid pQE-MANF. MANF was expressed in Escherichia coli (XL-blue, Agilent) and purified by immobilized-metal affinity chromatography on Ni-NTA Agarose columns (QIAGEN) under native conditions. Protein was eluted from the Ni-NTA columns with a buffer containing 250 mM imidazole. The elution buffer was exchanged to phosphate buffered saline (PBS) on Econo-Pac 10DG columns (Bio-Rad Laboratories). The purified rhMANF in PBS was stored at -80°C until use. The protein has an apparent size of ∼20 kDa after electrophoresis on acrylamide gel (Fig. 1 ).
8
Protein Labeling and Purification Protocol
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A solution of concentrated His6-SUMO-TEV-P42-G41C protein in buffer D was incubated for 20 min with a 10-fold molar excess of TCEP and flushed for 1 min with nitrogen gas (N2). A 10-fold molar excess of 5-carboxyfluorescein (5-FAM) maleimide (Thermo Scientific, Waltham, MA, USA) dissolved in dimethylsulfoxyde (DMSO) was added dropwise and the mixture was incubated overnight in the dark. A 10-fold molar excess of reduced glutathione was added and the solution was loaded on a PD10 desalting column (Econo-Pac® 10DG columns, Bio-Rad, Hercules, CA, USA) and eluted with buffer D. The protein was cleaved with the TEV protease at an approximate weight ratio of 100:2 (fusion protein/TEV) and overnight incubation at 4 °C to remove the tag. The labeling efficiency was assessed using liquid chromatography coupled to electrospray ionization mass spectrometry (ISBG facility, Grenoble). The labeled peptide P1–42, G41C-FAM was analyzed by SEC-MALLS (Multi-Angle Static Light Scattering—Dawn Heleos II, Wyatt) and the concentration was measured by on-line refractometry (Optilab T-rex refractometer, Wyatt).
9
Adenovirus PEGylation for Delivery
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Ad6-Luc was PEGylated as in ref. 35 (link). Following CsCl centrifugation, the preparation was desalted on EconoPac 10-DG columns (BioRad, Hercules, CA) into 0.5 M sucrose in PBS (136 mmol/l NaCl, 2.6 mmol/l KCl, 1.7 mmol/l KH2PO4, 10 mmol/l K2HPO4). 5 or 20 kDa succinimide-activated PEG (NOF America, White Plains, NY) was reacted with Ad6 at 10 mg/ml PEG for 1 hour at room temperature with rotation. Unreacted PEG was removed on a Sephadex G50 (GE Healthcare, Piscataway, NJ) size exclusion column. Mock-treated Ad5 was treated with no addition of PEG and treated in parallel.
10
Purification of PjTA Enzyme Variants
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Mutations
were introduced in vector pET-20b (+)-His-PjTA-R632 (link)via QuikChange mutagenesis
and confirmed by DNA sequencing (Eurofins Genomics). PjTA variants were produced in Escherichia coli BL21(DE3) cultivated in 200 mL of Terrific Broth (TB) medium with
100 μg/mL ampicillin. Expression was induced with IPTG at OD600 0.6, and cultivation was continued at 24 °C for 16
h. Cells were harvested by centrifugation, washed, and lysed by sonication
in 20 mM potassium phosphate, pH 7.5, containing 500 mM NaCl and 20
μM PLP. Centrifugation (36,000×g, 45 min,
4 °C) gave cell-free extract, loaded on a 5 mL HisTrap column.
Elution with a linear gradient of 0–500 mM imidazole in 20
mM potassium phosphate, pH 7.5, containing 500 mM NaCl and 20 μM
PLP, followed by desalting with Econo-Pac 10DG columns (Bio-Rad) gave
purified enzymes. Protein concentrations were determined with the
Bradford assay, and purified enzymes were stored in aliquots at −80
°C in 50 mM potassium phosphate, pH 8.0, with 20 μM PLP.
were introduced in vector pET-20b (+)-His-PjTA-R632 (link)via QuikChange mutagenesis
and confirmed by DNA sequencing (Eurofins Genomics). PjTA variants were produced in Escherichia coli BL21(DE3) cultivated in 200 mL of Terrific Broth (TB) medium with
100 μg/mL ampicillin. Expression was induced with IPTG at OD600 0.6, and cultivation was continued at 24 °C for 16
h. Cells were harvested by centrifugation, washed, and lysed by sonication
in 20 mM potassium phosphate, pH 7.5, containing 500 mM NaCl and 20
μM PLP. Centrifugation (36,000×g, 45 min,
4 °C) gave cell-free extract, loaded on a 5 mL HisTrap column.
Elution with a linear gradient of 0–500 mM imidazole in 20
mM potassium phosphate, pH 7.5, containing 500 mM NaCl and 20 μM
PLP, followed by desalting with Econo-Pac 10DG columns (Bio-Rad) gave
purified enzymes. Protein concentrations were determined with the
Bradford assay, and purified enzymes were stored in aliquots at −80
°C in 50 mM potassium phosphate, pH 8.0, with 20 μM PLP.
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