Hitrap protein a hp column

Manufactured by GE Healthcare

Sourced in United States, United Kingdom, Sweden

The HiTrap Protein A HP column is a pre-packed, ready-to-use column for the purification of antibodies and antibody fragments. The column is designed for high-performance affinity chromatography and utilizes Protein A ligand immobilized on a high-performance agarose matrix to capture and purify immunoglobulins from complex samples.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Protein A column (70 citations) HiTrap Protein A HP (25 citations) HiTrap HP column (20 citations) HiTrap Protein A (14 citations) HiTrap columns (11 citations)

77 protocols using hitrap protein a hp column

Glycosylated UL7 Protein Expression and Antibody Production

Check if the same lab product or an alternative is used in the 5 most similar protocols

To express UL7 in a glycosylated form comparable to that of the native protein, stable clones were made by transfection of HEK293 with pCMV6-UL7Fc and selection with neomycin (G418; Gibco-BRL). Stable clones with G418 resistance were obtained by trypsinization of colonies, followed by plating at limiting dilutions onto a 96-well plate. Ten single clones were then expanded and analyzed for UL7 secretion by the use of mouse Fc and an IgG enzyme-linked immunosorbent assay (ELISA) kit (MednaBio). The clone producing the largest amounts of fusion protein was cultured in EX-Cell 293 serum-free medium (Sigma-Aldrich). The supernatant containing the fusion protein was purified using a HiTrap protein A HP column (GE Healthcare) according to the manufacturer’s specifications.
To generate an anti-UL7 serum, the recombinant protein was used for rabbit immunization. The serum obtained by bleeding at 10 days after the fourth immunization was purified on an HiTrap protein A HP column (GE Healthcare) according to the manufacturer’s specifications. An isotype control was generated by purifying rabbit serum prior immunization. Fc and CEACAM1 recombinant proteins were purchased at R&D Systems.

MacManiman J.D., Meuser A., Botto S., Smith P.P., Liu F., Jarvis M.A., Nelson J.A, & Caposio P. (2014). Human Cytomegalovirus-Encoded pUL7 Is a Novel CEACAM1-Like Molecule Responsible for Promotion of Angiogenesis. mBio, 5(6), e02035-14.

+ Open protocol

+ Expand

Affinity Purification of can225IgG

Check if the same lab product or an alternative is used in the 5 most similar protocols

The can225IgG cell culture supernatant of the chosen clone 3A3 (subclone 3A3/14E1, respectively) was dialyzed before purification in 20 mmol/L sodium phosphate buffer (pH 7.0) overnight at 4°C under gentle stirring. Dialysis buffer was exchanged at least twice. For purification of the dialyzed supernatant, the Fast Protein Liquid Chromatography system (FPLC; ÄKTA; GE Healthcare Europe GmbH) was applied, using either a 1 mL HiTrap Protein A HP column (GE Healthcare), a 1 mL recombinant Protein A column (UNOsphere SUPrA; Bio-Rad Laboratories, Inc.), or a 5 mL HiTrap Protein A HP column (GE Healthcare). Furthermore, several smallscale purification attempts using Protein G Sepharose 4 Fast Flow beads (GE Healthcare) were performed before FPLC purification with HiTrap Protein G HP 5 mL column (GE Healthcare) was established, with a flow rate of 5 mL/min. To elute bound can225IgG, 0.1 mol/L Glycine–HCl buffer (pH 2.7) was applied. Upon elution, samples were immediately neutralized with 1 mol/L Tris–HCl (pH 9.0).

Singer J., Fazekas J., Wang W., Weichselbaumer M., Matz M., Mader A., Steinfellner W., Meitz S., Mechtcheriakova D., Sobanov Y., Willmann M., Stockner T., Spillner E., Kunert R, & Jensen-Jarolim E. (2014). Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients. Molecular cancer therapeutics, 13(7), 1777-1790.

+ Open protocol

+ Expand

Antibody scFv Diabody-Fc Fusion Protein Production

Check if the same lab product or an alternative is used in the 5 most similar protocols

The gene encoding the antibody scFv domain was cloned into a pFUSE-Fc expression vector (Cat. No. pfuse-hg1fc2, Invivogen, Hong Kong) to generate abEC1.1 as a diabody-Fc fusion protein (Wu et al., 2001 (link)) which comprises the entire Fc domain of human immunoglobulin G1 (IgG1) (Silverton et al., 1977 (link); Bujak et al., 2014 (link); Frenzel et al., 2016 (link)). In addition, the pFUSE-Fc expression vector was modified to generate a variant of the antibody with a murine Fc (abEC1.1m). For antibody production, a FreeStyle^TM 293-F cell line (Thermo Fisher Scientific, Cat. No. R79007), maintained in Freestyle 293 Expression Medium (Thermo Fisher Scientific, Cat. No. 12338026), was stably transfected with the abEC1.1 or abEC1.1m expression vector. Expressed antibodies were purified using HiTrap Protein A HP columns (GE Healthcare, Cat. No. 17-0403-03) with the ÄKTApurifier 100 system (GE Healthcare). After purification, the buffer was exchanged to PBS (pH 7.4) and the antibodies were kept in PBS at 4°C.

Ziraldo G., Buratto D., Kuang Y., Xu L., Carrer A., Nardin C., Chiani F., Salvatore A.M., Paludetti G., Lerner R.A., Yang G., Zonta F, & Mammano F. (2019). A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function. Frontiers in Physiology, 10, 392.

+ Open protocol

+ Expand

Transient Transfection and Antibody Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Constructs were transiently transfected in HEK293-EBNA1-6E cells (NRC) or Expi293F cells (Thermo Fisher) at a density of 1.2-1.6 million cells/mL or 3 million cells/mL, respectively, using 25 kDa linear polyethylenimine (PEI) at a 3:1 PEI:DNA ratio in OptiMEM reduced serum medium (Gibco), with a heavy chain: light chain ratio of 1:1. HEK293-EBNA1-6E cells were maintained in Freestyle 293 Expression Medium (Gibco) and were supplemented with 0.5% tryptone 24-36 hours after transfection. Expi293F cells were maintained in Expi293 Expression Medium (Thermo Fisher). Cell supernatants were harvested after 96 hours and filtered through 0.45-μM filters (Millipore). Antibodies were purified via HiTrap Protein A HP columns (GE Healthcare) run at a flow rate of 0.5-1 mL/min on Äkta Start protein purification system (GE Healthcare). Antibodies were eluted using 0.1M glycine pH 2, dialyzed with 3 changes of PBS pH 7.4 using Slide-A-Lyzer-G2 10K dialysis cassettes (Thermo Fisher), and concentrated using 30,000 kDa molecular weight cutoff polyethersulfone membrane spin columns (Pierce). Final concentrations of purified antibodies were quantified with Nanodrop 2000 (Thermo Fisher).

Nielsen S.C., Yang F., Jackson K.J., Hoh R.A., Röltgen K., Jean G.H., Stevens B.A., Lee J.Y., Rustagi A., Rogers A.J., Powell A.E., Hunter M., Najeeb J., Otrelo-Cardoso A.R., Yost K.E., Daniel B., Nadeau K.C., Chang H.Y., Satpathy A.T., Jardetzky T.S., Kim P.S., Wang T.T., Pinsky B.A., Blish C.A, & Boyd S.D. (2020). Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2. Cell Host & Microbe, 28(4), 516-525.e5.

+ Open protocol

+ Expand

Immunoglobulin Purification from Synovial Fluid

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunoglobulins from synovial fluid were purified using HiTrap Protein A HP columns (GE Healthcare). Briefly, samples of synovial fluid from two patients with rheumatic inflammatory joint disease diluted 1:4 in PBS were loaded onto 1 mL columns. Bound immunoglobulins were thereafter eluted with 0.1 M sodium citrate (pH 3.0). The study was approved by the Committee of Ethics at the University of Bergen (nr. 242.06). All patients gave their informed consent.

Koro C., Bielecka E., Dahl-Knudsen A., Enghild J.J., Scavenius C., Brun J.G., Binder V., Hellvard A., Bergum B., Jonsson R., Potempa J., Blom A.M, & Mydel P. (2014). Carbamylation of immunoglobulin abrogates activation of the classical complement pathway. European Journal of Immunology, 44(11), 3403-3412.

+ Open protocol

+ Expand

Immunoglobulin Purification from Synovial Fluid

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunoglobulins from synovial fluid were purified using HiTrap Protein A HP columns (GE Healthcare). Briefly, samples of synovial fluid from two patients with rheumatic inflammatory joint disease diluted 1:4 in PBS were loaded onto 1 ml columns. Bound immunoglobulins were thereafter eluted with 0.1 M sodium citrate (pH 3.0). The study was approved by the Committee of Ethics at the University of Bergen (nr. 242.06). All patients gave their informed consent.

+ Open protocol

+ Expand

Purification and Quantification of Anti-GM-CSF Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human mAbs and total IgG from PAP sera were purified by protein A or protein G chromatography (GE Healthcare). Total IgG from healthy donors were purified using the HiTrap Protein A HP columns (GE Healthcare) and concentrated using Amicon Ultra filter units (100 K, Millipore). Total GM-CSF antibodies were affinity-purified from PAP sera using magnetic beads (Invitrogen) conjugated with human GM-CSF. Total IgGs were quantified using ELISA plates coated with anti-human IgG (SouthernBiotech) using Certified Reference Material 470 (ERMs-DA470, Sigma-Aldrich) as standard. Binding to GM-CSF was tested by ELISA using 384-well SpectraPlates (PerkinElmer) for primary screenings or 96-well MaxiSorp plates (Nunc) for any following test. Briefly, ELISA plates were coated with 1 μg ml⁻¹ of recombinant human GM-CSF (Gentaur), blocked with 1% BSA and incubated with titrated antibodies, followed by AP-conjugated anti-human IgG secondary antibodies (SouthernBiotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. EC50 (ng ml⁻¹) was calculated for every sample by nonlinear regression analysis using the GraphPad Prism 5 software.

Piccoli L., Campo I., Fregni C.S., Rodriguez B.M., Minola A., Sallusto F., Luisetti M., Corti D, & Lanzavecchia A. (2015). Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nature Communications, 6, 7375.

+ Open protocol

+ Expand

Purification of Anti-CD8 Monoclonal Antibody

Check if the same lab product or an alternative is used in the 5 most similar protocols

The OX-8 cell line (Health Protection Agency Culture Collections) producing a murine anti-CD8 monoclonal antibody was grown in CD Hybridoma Medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM l-glutamine and 2.8 mg/L gentamicin (PAA Laboratories GmbH) for 10 to 12 days. The medium was centrifuged, and the supernatant was collected and stored at −20°C. The antibodies were purified utilizing HiTrap™ Protein A HP columns (GE Healthcare Life Sciences, Uppsala, Sweden), and the eluted antibodies were transferred to phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) by repeated centrifugation using an Amicon-15 filter unit (molecular weight cutoff 30,000, Millipore, Billerica, MA, USA). The purity of the antibody solution was analyzed by bioanalyzer and a Protein 230 plus kit (Agilent Technologies, Santa Clara, CA, USA). The protein concentration was determined using the BCA (bicinchoninic acid) standard assay (Sigma-Aldrich, St. Louis, MO, USA). The purified antibody solution was stored at −20°C.

Elgström E., Eriksson S.E., Ohlsson T.G., Nilsson R, & Tennvall J. (2015). Role of CD8-positive cells in radioimmunotherapy utilizing 177Lu-mAbs in an immunocompetent rat colon carcinoma model. EJNMMI Research, 5, 3.

+ Open protocol

+ Expand

Transient Transfection and Antibody Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Constructs were transiently transfected in HEK293-EBNA1–6E cells (NRC) at a density of 1.2–1.6 million cells/mL using 25 kDa linear polyethylenimine (PEI) at a 3:1 PEI:DNA ratio in OptiMEM reduced serum medium (Gibco), with a heavy chain: light chain ratio of 1:1. Cells were maintained in Freestyle 293 Expression Medium (Gibco) and were supplemented with 0.5% tryptone 24–36 hours after transfection. Cell supernatants were harvested after 96 hours and filtered through 0.45-μM filters (Millipore). Antibodies were purified via HiTrap Protein A HP columns (GE Healthcare) run at a flow rate of 0.5–1 mL/min on Äkta Start protein purification system (GE Healthcare). Antibodies were eluted using 0.1M glycine pH 2, dialyzed with 3 changes of PBS pH 7.4 using Slide-A-Lyzer-G2 10K dialysis cassettes (Thermo Fisher), and concentrated using 30,000 kDa molecular weight cutoff polyethersulfone membrane spin columns (Pierce). Final concentrations of purified antibodies were quantified with Nanodrop 2000 (Thermo Fisher).

Nielsen S.C., Yang F., Jackson K.J., Hoh R.A., Röltgen K., Stevens B., Lee J.Y., Rustagi A., Rogers A.J., Powell A.E., Najeeb J., Otrelo-Cardoso A.R., Yost K.E., Daniel B., Chang H.Y., Satpathy A.T., Jardetzky T.S., Kim P.S., Wang T.T., Pinsky B.A., Blish C.A, & Boyd S.D. (2020). Human B cell clonal expansion and convergent antibody responses to SARS CoV-2. bioRxiv.

+ Open protocol

+ Expand

Fc-Fusion Protein Expression Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

To obtain expression vector for fusion proteins of mouse GP2, human GP2, mouse uromodulin (Umod) or mouse PrP^C with the Fc segment of human IgG1 (mGP2-Fc, hGP2-Fc, Umod-Fc and PrP^C-Fc), complementary DNAs (cDNAs) corresponding to the extracellular domains of each protein were amplified by PCR using primer sets listed in Supplementary Table 1. The cDNA fragments were inserted into the pcDNA3 expression vector (Invitrogen) containing a fragment encoding the Fc segment of human IgG1 to generate Fc-fusion proteins21 (link)22 (link). Human embryonic kidney 293T (HEK293T) cells were transiently transfected with the expression vectors using Lipofectamine 2000 (Invitrogen), and then cultured for several days. The Fc-fusion proteins secreted into the culture supernatant were purified on HiTrap protein A HP columns (GE Healthcare).

Matsumura T., Sugawara Y., Yutani M., Amatsu S., Yagita H., Kohda T., Fukuoka S.I., Nakamura Y., Fukuda S., Hase K., Ohno H, & Fujinaga Y. (2015). Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity. Nature Communications, 6, 6255.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!