Hitrap protein l column

Manufactured by GE Healthcare

Sourced in Germany

The HiTrap Protein L column is a prepacked affinity chromatography column designed for the purification of immunoglobulins (Igs) from various sample sources. The column utilizes Protein L, a bacterial cell wall protein that binds to the light chains of most Igs, allowing for the capture and isolation of Igs without the need for prior knowledge of the Ig subclass or antigen specificity.

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Lab products found in correlation

Hitrap, by Cytiva (8 mentions) Expi293f cells, by Thermo Fisher Scientific (2 mentions) Expi293 expression medium, by Thermo Fisher Scientific (2 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions) Precision plus protein dual color standard, by Bio-Rad (1 mentions) Immobilized papain, by Thermo Fisher Scientific (1 mentions) Hitrap protein a column, by GE Healthcare (1 mentions) Akta fplc system, by GE Healthcare (1 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions) Hitrap mabselect sure column, by GE Healthcare (1 mentions) Mabselect sure, by Cytiva (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Amicon ultra 10k device, by Merck Group (1 mentions) Superdex 200 10 300 gl column, by GE Healthcare (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Plasmid midi kit, by Qiagen (1 mentions) Zeocin, by Thermo Fisher Scientific (1 mentions) Calf intestinal alkaline phosphatase, by Promega (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)

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HiTrap columns (11 citations) HiTrap protein L (2 citations) Protein L column (2 citations)

8 protocols using hitrap protein l column

Expressing and Purifying scFv Antibody Fragments

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The scFv cassette was batch-cloned as NcoI/NotI fragments from the phagemids into pFKPEN56 (link), placing the scFvs in-frame with c-myc and his-tags, and transformed into E. coli XL1-Blue (Stratagene). Individual clones were inoculated and expressed in 96-deep well plates using 400 μl culture medium for screening experiments or in either 10 ml or 200 ml cultures for larger-scale expressions. These were performed essentially as described, except that 2x YT was used as culture medium and 0.1 mM IPTG was used for induction of protein expression in small scale expressions56 (link). For 96-deep well expression 50 μl was used to re-inoculated new cultures after overnight growth. Single clones identified by screening were sequenced by GATC Biotech.
For purification of OMV-reactive scFvs, periplasmic samples were diluted 4x with PBS and pH adjusted to 7.4, before affinity purification using a HiTrap Protein L column (GE Healthcare). Proteins were eluted with 0.1 M Glycine-HCl pH 2.7 and neutralized by addition of 1 M Tris-HCl pH 8. For size exclusion chromatography, Superdex 200 Increase 10/300 GL run in PBS supplemented with 150 mM NaCl was used. pH was adjusted according to the pI of the individual samples.

Høydahl L.S., Nilssen N.R., Gunnarsen K.S., Pré M.F., Iversen R., Roos N., Chen X., Michaelsen T.E., Sollid L.M., Sandlie I, & Løset G.Å. (2016). Multivalent pIX phage display selects for distinct and improved antibody properties. Scientific Reports, 6, 39066.

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Purification of His-tagged Proteins

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Cultures were centrifuged at 6500xg at 4 °C (Avanti, Beckman Coulter). Protein-containing supernatant was incubated with His-Pur Ni-NTA resin (Thermo Fisher) for 2 h at 4 °C, mixing gently. The mixture was loaded onto a 10 ml vacuum column (Thermo Fisher) and purified according to the manual’s protocol. 1X phosphate-buffered saline (PBS) with 25 mM imidazole, pH 7.4 and PBS with 500 mM imidazole, pH 7.4 were used as a wash and elution buffers, respectively. Purified proteins were buffer-exchanged^{53 (link)} into PBS (pH 7.4) through membrane filtration (Amicon® Ultra-4 Centrifugal Filter Units, MWCO 10 kDa, Merck). Protein samples were then loaded onto the HiTrap™ Protein L column (GE Healthcare) to achieve better purity (> 95%). Protein purities were confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Precision Plus Protein™ Dual Color standard was used as a marker (Bio-Rad). Protein concentration was determined by NanoDrop 2000 (absorbance at 280 nm).

Arslan M., Karadag M., Onal E., Gelinci E., Cakan-Akdogan G, & Kalyoncu S. (2022). Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv. Scientific Reports, 12, 5449.

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Antibody Purification and Fab Fragmentation

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Antibody was purified from ascites by HiTrap Protein A column (GE Healthcare) according to manufacturer’s instructions. Briefly, ascites was diluted in 1× phosphate buffer saline (PBS) and injected in pre-equilibrated Protein A column. The whole antibody was eluted from the column using 25 mM glycine (pH 2.2) elution buffer and then subjected to papain cleavage according to manufacturer’s instructions. In brief, whole antibody was incubated with immobilized papain (Thermo Scientific) in digestion buffer (20 mM sodium phosphate (pH 7), 10 mM EDTA, 20 mM cysteine) overnight at 37 °C which was followed by Fab purification by HiTrap Protein L column (GE Healthcare) according to manufacturer’s instructions.

Barnwal B., Mok C.K., Wu J., Diwakar M.K., Gupta G., Zeng Q., Chow V.T., Song J., Yuan Y.A, & Tan Y.J. (2015). A monoclonal antibody binds to threonine 49 in the non-structural 1 protein of influenza A virus and interferes with its ability to modulate viral replication. Antiviral Research, 116, 55-61.

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Expi293F Cell-Based Antibody Production

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Expi293F cells (Life Technologies Corporation, Carlsbad, CA) were cultured in Expi293 Expression Medium (Life Technologies Corporation, Grand Island, NY). Expi293F cells were transfected using an Expifectamine 293 Transfection kit (Gibco, Carlsbad, CA), following the manufacturer's protocol. Cells were cultured for 5 days after transfection by shaking at 250 rpm in a 37 °C incubator with 8% CO₂. IgG and antibody fragments were harvested from supernatant by centrifugation at 3,000 ×g for 20 min and filtered using a 0.45 μm filter. scFv-C_H3 was purified using GE Healthcare AKTA FPLC system with HiTrap Protein L column (GE healthcare, Sweden). scFv-Fc and IgG1 were purified using HiTrap MabSelect SuRe column (GE healthcare, Sweden). Further purification was performed as described for antibody fragments expressed in E. Coli. Protein sequences of antibody fragments are listed in Table S1.

El-Sayed A., Bernhard W., Barreto K., Gonzalez C., Hill W., Pastushok L., Fonge H, & Geyer C.R. (2018). Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts. Theranostics, 8(17), 4856-4869.

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Purification of HJ8.5 scFv Antibody

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HJ8.5 monoclonal antibody was raised by immunizing tau-knockout mice with htau as described previously (Yanamandra et al., 2013 (link)). This antibody recognizes amino acids 25–30 of the full-length human 1N4R sequence (Yanamandra et al., 2013 (link)). After sequencing, the variable regions were cloned and combined in one plasmid connected by a (GGGS)₁-linker. A secretory signal peptide was added at the N terminus and an HA-tag at the C terminus. The different linker sequences were introduced by site-directed mutagenesis. For purification, Expi293F cells (Gibco) were grown in Expi293 Expression Medium (Gibco) to a density of 10⁶ cells/ml before they were transiently transfected with Lipofectamine 2000 (Invitrogen). The scFvs were affinity-purified from supernatant 4 d later by using a HiTrap Protein L column (GE Healthcare) according to the manufacturers’ instructions. After elution, the scFvs were dialyzed in PBS and concentrated with an Amicon Ultra 10K device (EMD Millipore).

Ising C., Gallardo G., Leyns C.E., Wong C.H., Jiang H., Stewart F., Koscal L.J., Roh J., Robinson G.O., Remolina Serrano J, & Holtzman D.M. (2017). AAV-mediated expression of anti-tau scFvs decreases tau accumulation in a mouse model of tauopathy. The Journal of Experimental Medicine, 214(5), 1227-1238.

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Papain-Digested Fab Purification

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Fabs were generated by papain digestion using crystallized papain (Sigma-Aldrich; Cat #1495005) in 50 mM NaPO₄, 2 mM EDTA, 10 mM L-cysteine, pH 7.4 for 30–60 min at 37°C at a 1:100 enzyme:IgG ratio. Fab and partially cleaved IgG were applied on 1-mL HiTrap Protein L column (GE Healthcare Life Science; Cat #29048665). After loading, the column was washed with 10 column volumes of 10 mM NaPO₄ and 150 mM NaCl at pH 7.0. The protein was eluted with 100 mM Na citrate, pH 2.5, then immediately neutralized using 2 M Tris pH 8.0. The elution fractions were pooled and dialyzed into 10 mM Hepes and 150 mM NaCl at pH 7.4. Fabs were further purified by SEC using a Superdex 200 10/300 GL column (GE Healthcare Life Sciences; Cat #17517501) in 10 mM Hepes and 150 mM NaCl at pH 7.4.

Tanaka S., Olson C.A., Barnes C.O., Higashide W., Gonzalez M., Taft J., Richardson A., Martin-Fernandez M., Bogunovic D., Gnanapragasam P.N., Bjorkman P.J., Spilman P., Niazi K., Rabizadeh S, & Soon-Shiong P. (2022). Rapid identification of neutralizing antibodies against SARS-CoV-2 variants by mRNA display. Cell Reports, 38(6), 110348.

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Recombinant Protein Expression in P. pastoris

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E. coli XL1-Blue Chemically Competent Cells (Agilent Technologies, Santa Clara, CA, USA) were employed for the propagation of plasmids, and P. pastoris X-33 strain (Life Technologies, Carlsbad, CA, USA) was used for scFv and BirA enzyme expression. P. pastoris expression vectors pPICZαB and pPIC6αA were purchased from Life Technologies. Restriction enzymes PstI, NotI, XbaI, EcoRI and SacI, calf intestinal alkaline phosphatase, T4 DNA ligase, and GoTaq DNA Flexi Polymerase were purchased from Promega (Madison, WI, USA). Q5 High-Fidelity DNA Polymerase was purchased from New England Biolabs (Hitchin, UK). Synthetic oligonucleotides were purchased form Sigma-Aldrich (Gillingham, UK). Plasmid purification kit (QIAGEN Plasmid Midi Kit), PCR product purification kit (QIAquick PCR Purification Kit) and gel extraction kit (QIAquick Gel Extraction Kit) were purchased from Qiagen (Cologne, Germany). HiTrap Protein L Column was purchased from GE Healthcare Life Sciences (München, Germany). Selection antibiotic Zeocin was purchased from Life Technologies, and blasticidin was purchased from InvivoGen (Toulouse, France).
Peptone, tryptone, yeast extract, and European bacteriological agar were purchased from Pronadisa (Madrid, Spain). Methanol was purchased from Fisher Scientific (Loughborough, UK). Other chemicals were purchased from Sigma-Aldrich unless otherwise stated.

de la Cruz S., Alcocer M., Madrid R., García A., Martín R., González I, & García T. (2016). Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris. Journal of biotechnology, 227.

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Biotinylated scFv Purification Protocol

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The supernatant containing biotinylated scFv was filtered through a 0.4 μm membrane filter (Millipore, Darmstadt, Germany) and loaded onto a 1x1 mL HiTrap protein L column (GE Healthcare Life Sciences) attached to an ÄKTA purifier FPLC system (GE Healthcare, Sweden). Chromatography was performed as described by Rouet et al., 2012 with several modifications. Briefly, 300 mL of supernatant was loaded onto the column previously equilibrated with 10 mL of PBS buffer (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4). The column was then washed with 20 mL of PBS, and the biotinylated scFv eluted with 10 mL of 0.1 M glycine-HCl (pH 2.7). Fractions showing OD 280 above 0.05 were manually collected in 1.5 mL microcentrifuge tubes prefilled with 400 mL of 200 mM Tris-HCl (pH 8.0).
Flow rate was maintained at 1 mL min -1 . The entire process was repeated with the remaining 300 mL of supernatant.
Recovered fractions were pooled and dialyzed against PBS buffer employing Amicon Ultra-15 Centrifugal Filter Units (Millipore) with a MWCO of 10 kDa. Protein concentration was measured in a Nanodrop (Thermo Scientific, Waltham, MA, USA), adjusted to 2 mg mL -1 of total protein, and stored in 100 µL aliquots at -80 ºC until further use.

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