Monoclonal mouse antibody specific for human EGFR (IgG2a, clone Mab 108) was produced from hybridoma HB-9764 obtained from ATCC (Manassas, VA). Hybridoma cells were grown in RPMI media containing 10% fetal bovine serum, 5% penicillin/streptomycin, 5% L-glutamine, and 1% Sodium Pyruvate and subcultured by dilution of the non adherent cells. For antibody production, 500 mL hybridoma cell cultures were grown for 7 days from an initial seeding density of 5 × 105 cells/mL. The media was collected, centrifuged at 3000 × g for 10 min to remove cell debris, concentrated and buffer exchanged into PBS using a Labscale tangential flow filtration system fitted with a Pellicon XL cassette (both from Millipore, Billerica, MA), and purified using a HiTrap Protein G column (GE Healthcare, Piscataway, NJ).
Labscale tangential flow filtration system
Manufactured by Merck Group
Sourced in United States, Germany
The Labscale tangential flow filtration system is a laboratory-scale equipment designed for the separation and purification of molecules and particles in liquid samples. It utilizes a cross-flow filtration technique, where the feed solution flows parallel to the membrane surface, allowing for the selective retention of desired components while the permeate, or filtered liquid, is collected. The system is capable of handling a range of sample volumes and can be used for various applications in the fields of biotechnology, pharmaceutical research, and process development.
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Lab products found in correlation
7 protocols using labscale tangential flow filtration system
1
Purification of Anti-EGFR Monoclonal Antibody
2
Antioxidant Activity of S. limacinum Hydrolysates
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The S. limacinum hydrolysates (SLHs) were dissolved in distilled water and then fractionated through an ultrafiltration membrane system (Labscale tangential-flow filtration system, Milipore, Billerica, MA, USA) with a molecular weight cutoff (MWCO) of 50, 10, and 5 kDa, respectively. Three fractions, SLH-I (<50 kDa), SLH-II (<10 kDa), and SLH-III (<5 kDa) were obtained. All samples were lyophilized and subjected to antioxidant activity test.
3
Production and Characterization of PI-Tf Fusion Protein
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Example 1
PI-Tf Recombinant Fusion Protein Expression and Characterization
Preproinsulin sequence (NM_000207) (SEQ ID NO: 1) fused in frame with Tf sequence (NM_001063) (SEQ ID NO: 2) was engineered into pcDNA3.1 (+) expression vector (Invitrogen, CA) by molecular cloning methods (
4
Isolating Antihypertensive Peptides from Seahorse
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Peptides were extracted and purified from Hippocampus abdominalis alcalase hydrolysate (HA) following the procedures reported by Kim et al. In brief, dried seahorse (5 g) was mixed with 100 mL of distilled water containing 10 mg of alcalase enzyme and then extracted under optimal conditions (pH 4.5, 37 °C) for 24 h. After hydrolyzation for 24 h, alcalase-assisted hydrolysates were centrifuged (12,902× g rpm, 4 °C, 15 min) and the supernatant was filtered followed by heat inactivation (105 °C, 10 min). The hydrolysates were then subjected to separating membranes and the hydrolysates were then fractionated using an ultrafiltration system (Labscale Tangential Flow Filtration System, Millipore, Merck KGaA, Darmstadt, Germany) equipped with molecular weight cutoff (MWCO) membranes. The hydrolysates were fractionated using two MWCO membranes in decreasing sizes (10 kDa and 5 kDa). Three fractions (>10 kDa, HA-Ι; 5–10 kDa, HA-ΙΙ; <5 kDa, HA-ΙΙΙ) were obtained. Of the fractions collected, the HA-III (<5 kDa) fraction was loaded onto a Sephadex G10 open column to separate the active peptide. Ultimately, four peptide fractions (HA-ΙΙΙ-a, HA-ΙΙΙ-b, HA-ΙΙΙ-c, and HA-ΙΙΙ-d) were obtained, and IGTGIPGIW, the potential antihypertensive peptide of interest, was isolated from HA-ΙΙΙ-b.
5
Inactivation and Concentration of African Horse Sickness Virus
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Marc-145 cells were incubated with the master seed virus inoculum in MEM with 5% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. The AHSV infectivity titers were calculated before concentration as ≥1 × 107 plaque-forming units/mL. After 7 days of infection, virus particles and cell suspension were harvested by freeze-thawing 3 times. After centrifugation, AHSV was inactivated by the addition of 0.1% formalin (Merck, Germany) and incubated for 2 weeks. The virus was concentrated 10 times and formalin removed using the Labscale™ Tangential Flow Filtration System (Millipore, USA) for prototype vaccine production. The inactivated virus solution was tested for residual viable virus by passaging in MARC-145 cells in T75 tissue culture flasks. The AHSV’s residual viral RNA was detected in the final passages by PCR. All inactivated viral suspensions were stored at 2–8°C until formulation with an adjuvant [14 , 15 ].
6
Purification of Recombinant Protein from Yeast Fermentation
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After cultivation, cells were removed from the induced yeast fermentation broth by centrifugation at 6,000 × g, 4 °C for 40 min. About 500 mL fermentation clear broth was subjected to microfiltration (0.45 μm), concentrated by ultrafiltration on a Labscale™ Tangential Flow Filtration System (Merck Millipore, German) equipped with a 30 kDa cut off module (Pellicon® XL, 50 cm2, Millipore, Germany). The concentrated broth was diluted and concentrated twice with Ni–NTA buffer A. Samples were centrifugated at 10,000 × g, 4 °C for 30 min to remove deactivated proteins, then further purified by Ni2+-affinity chromatography (Ren et al. 2020 (link)) and flash frozen in liquid nitrogen, stored at ‒80 °C for further analysis.
7
Isolation and Purification of Lytic Phage AH67C600_Q9
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The lytic phage AH67C600_Q9 was isolated from a wastewater sample obtained from a pig farm sewage treatment system through additional pretreatment steps [27 (link),28 (link)]. In brief, the sewage samples were concentrated by an ultrafiltration device (Millipore Labscale tangential flow filtration System, Merck KGaA, Darmstadt, Germany) and then filtered through a 0.22 µm membrane (Millex-GP, Millipore, Burlington, MA, USA) [29 (link)]. The filtrate was incubated with logarithmic phase cells of E. coli AH67C600 overnight at 37 °C with shaking at 160 rpm. The mixture was centrifuged and filtered as per above and used to inoculate new bacterial cultures to enrich the primary phage preparation. The final filtrates were plaque-purified using the double-layer soft agar plate method [30 (link),31 (link)]. Single plaques were selected and diluted in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 7.5) and stored at 4 °C. The single phage was plaque-purified three times by E. coli C600 before use and standardized titers in the range of 107–109 plaque forming units (PFU) per mL were used for follow-up experiments.
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