After 48 h of incubation, the bacterial culture broths were centrifuged at 8000 g for 20 min at 4°C. The cell‐free supernatant was 20‐fold concentrated at 70°C for 24 h and centrifuged at 100,000 g for 1 h at 4°C to remove the precipitate. The resulting supernatant was then filtered through a 0.2 μm pore‐size membrane (Millipore). The filtrate was fractionated through stepwise ultrafiltration using 10 and 1 kDa cutoff discs (Millipore) to yield high molecular weight (HMW, >10 kDa), middle molecular weight (MMW, 1–10 kDa), and low molecular weight (LMW, <1 kDa) fractions. The fractions were lyophilized and stored at −70°C until use. Each fraction (100 mg) was dissolved in 20 mM Tris–HCl (pH 7.0) and loaded onto a Superdex 30 prep‐grade (pg) column (200 ml) pre‐equilibrated with the same buffer in a Biologic Duo‐Flow FPLC system (Bio‐Rad) at a rate of 1.5 ml/min. Eluents were monitored by measuring the absorbance at 210 nm, and each peak fraction was pooled, lyophilized, and stored at −20°C until use. Based on the standard curve obtained using proteins/peptides of known sizes (Figure S1 ), 1–10 kDa peptide fractions were pooled and used for further analysis.
Biologic duoflow fplc system
Manufactured by Bio-Rad
Sourced in United States
The BioLogic DuoFlow FPLC system is a versatile fast protein liquid chromatography (FPLC) instrument designed for protein purification and analysis. It features a modular design and supports a range of chromatography techniques, including ion exchange, size exclusion, and affinity chromatography. The system is capable of handling a wide variety of sample types and volumes, and can be customized to meet specific research or laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Ktapure fplc system, by Cytiva (2 mentions)
Ultra 15 centrifugal filter units, by Cytiva (2 mentions)
Superdex 200 pg 16 60 column, by GE Healthcare (2 mentions)
Model 705 sonic dismembrator, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
0.2 μm pore size membrane, by Merck Group (1 mentions)
Ultracel 3k, by Merck Group (1 mentions)
Phosphocellulose p 11, by Cytiva (1 mentions)
Unos6, by Bio-Rad (1 mentions)
Enrich sec 70 column, by Bio-Rad (1 mentions)
Infinity cholesterol reagent, by Thermo Fisher Scientific (1 mentions)
Superose 6 column, by Cytiva (1 mentions)
Qiaprep miniprep kit, by Qiagen (1 mentions)
Top10 cells, by Thermo Fisher Scientific (1 mentions)
8 protocols using biologic duoflow fplc system
1
Peptide Fractionation and Purification
2
Purification of RNase from B. licheniformis
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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.
3
Purification of Recombinant DmmarA Protein
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The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml−1). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min−1 for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min−1. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).
4
Purification of Recombinant DmmarA Protein
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The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml−1). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min−1 for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min−1. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).
5
Elution Profiling of Polypeptide Fibrillation
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Elution profiling of polypeptides
by SEC was monitored using an Quadtech detector in a Biologic Duoflow
FPLC system (BioRad laboratories) at λ = 280 nm. Lyophilized
polypeptides were dissolved in 5 mL of Milli-Q water to a concentration
of 0.6 mM. 0.5 mL of this solution was syringe filtered and loaded
on the Enrich SEC-70 column (BioRad laboratories) equilibrated in
50 mM sodium phosphate buffer (pH = 5.5). The remaining polypeptides
in a polypropylene tube were shaken at 600 rpm incubated at 58 °C.
0.5 mL of aliquots were taken out at 12, 36, and 84 h of fibrillation
and subjected to size-exclusion profiling after centrifugation at
18 000 rpm in a microcentrifuge tube and filtering the solution
through a 0.45 μm PES membrane syringe filter. The SEC-70 column
was calibrated using lysozyme (14.5 kDa) and wt protein GB1(6.2 kDa)
in phosphate buffer at pH = 5.5.
by SEC was monitored using an Quadtech detector in a Biologic Duoflow
FPLC system (BioRad laboratories) at λ = 280 nm. Lyophilized
polypeptides were dissolved in 5 mL of Milli-Q water to a concentration
of 0.6 mM. 0.5 mL of this solution was syringe filtered and loaded
on the Enrich SEC-70 column (BioRad laboratories) equilibrated in
50 mM sodium phosphate buffer (pH = 5.5). The remaining polypeptides
in a polypropylene tube were shaken at 600 rpm incubated at 58 °C.
0.5 mL of aliquots were taken out at 12, 36, and 84 h of fibrillation
and subjected to size-exclusion profiling after centrifugation at
18 000 rpm in a microcentrifuge tube and filtering the solution
through a 0.45 μm PES membrane syringe filter. The SEC-70 column
was calibrated using lysozyme (14.5 kDa) and wt protein GB1(6.2 kDa)
in phosphate buffer at pH = 5.5.
6
Purification of pAcFRS from BL21(DE3)
7
Plasma Lipid and Cholesterol Analysis
8
Isolation and Purification of Single-Domain Antibodies
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Plasmid from individual colonies of positive SEB binders identified by monoclonal phage ELISA was purified from overnight cultures using the QIAprep Miniprep Kit (Qiagen) and sequenced by Eurofins MWG Operon (www.operon.com ). Genes coding for individual sdAbs from the isolated plasmids were cloned into expression vector pECAN45 by restriction digestion with SfiI and ligated with T4 DNA ligase. Expression plasmids were maintained in Top10 cells (Life Technologies, Grand Island, NY, USA) and transformed into Rosetta (DE3) cells (Novagen) for protein expression. In order to improve the yield of three of the sdAbs (S222-A2, H9-B11, and H9-H11), the genes were amplified by PCR to remove the C-terminal hinge region and cloned into the NcoI and NotI sites of expression vector pET22b. Expression and purification of individual sdAbs was performed as described previously [25 (link)]. Purification of sdAbs isolated from the periplasmic space was accomplished by immobilized metal affinity chromatography (IMAC) followed by FPLC size exclusion chromatography using a BioLogic Duo Flow FPLC system (BioRad, Hercules, CA, USA) equipped with a Sephadex G75 column. The concentration of purified protein solutions was determined on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
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