Human leukocyte antigen (HLA) antibodies specific to HLA-A2 (BB7.2 [HB-82; ATCC] [20 (link)] and pan-HLA class I (W6/32 [HB-95; ATCC] [21 (link)] were purified from hybridoma cells. Secreted monoclonal antibodies, BB7.2 (anti-HLA-A2) and W6/32 (pan-HLA class I), were harvested from spent media and purified using Protein A Sepharose (PAS, CaptivA®, Repligen, Waltham, MA, USA) using a Profinia purification system (Biorad, Hercules, CA, USA). Next, an affinity matrix wherein 10 mg/mL of BB7.2 (anti-HLA-A2) and W6/32 (pan-HLA class I) were crosslinked to 1 mL of PAS resin were prepared for each of the transfected SaOS cell line, as previously described [22 (link)].
Profinia purification system
Manufactured by Bio-Rad
Sourced in United States, Australia
The Profinia purification system is a fully automated chromatography system designed for protein purification. It enables the efficient and reproducible purification of a wide range of proteins. The Profinia system provides a streamlined workflow, simplifying the purification process and reducing hands-on time.
Automatically generated - may contain errors
Lab products found in correlation
Hiload 16 60 superdex 75 column, by GE Healthcare (1 mentions)
Cell disruptor, by Constant Systems (1 mentions)
Quickchange lightning, by Agilent Technologies (1 mentions)
Kta fast protein liquid chromatography system, by GE Healthcare (1 mentions)
Q5 site directed mutagenesis, by New England Biolabs (1 mentions)
Oneshot bl21 de3 chemically competent e coli cells, by Thermo Fisher Scientific (1 mentions)
Ultrasonication, by Thermo Fisher Scientific (1 mentions)
Imac column, by Bio-Rad (1 mentions)
Hitrap streptavidin hp column, by GE Healthcare (1 mentions)
6 protocols using profinia purification system
1
Purification and Immobilization of HLA Antibodies
2
Purification of Mutant Human PKG Iα
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A DNA construct containing His-tagged human PKG Iα (79–212) R177Q was cloned into pQTEV and transformed into TP2000 E. coli cells (Kim et al., 2015 (link); Roy and Danchin, 1982 (link)). Cells were grown at 37°C until OD600 of 0.6 and induced with 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Cells were then grown for an additional 10 hr at 25°C, harvested by centrifugation. Cells were suspended in Buffer A (50 mM potassium phosphate, 500 mM NaCl, 1 mM β-mercaptoethanol [pH 7.5]) and lysed with a cell disruptor (Constant Systems, Daventry Northants, UK). The protein was purified with BioRad IMAC resin on a BioRad Profinia purification system and eluted with Buffer A containing 300 mM imidazole. The elution fraction protein sample was incubated with TEV protease at 4°C overnight for His-tag removal. A second nickel affinity chromatography step was performed to remove TEV protease and flow-through fractions were collected. Protein was further purified with gel filtration on a Hi-load 16/60 Superdex-75 column (GE Healthcare) in 25 mM Trizma (pH 7.5), 150 mM NaCl, and 1 mM TCEP–HCl.
3
Purification of pET28b and pEG202 Laforin Mutants
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All pET28b and pEG202 laforin mutants were generated by site-directed mutagenesis (QuickChange Lightning, Agilent; Q5 Site-Directed Mutagenesis, New England BioLabs; GENEWIZ custom Site-Directed Mutagenesis). All pET28b mutants were expressed in OneShot BL21(DE3) Chemically Competent E. coli cells (ThermoFisher) and purified using immobilized metal affinity chromatography and a Profinia Purification System (BioRad) and size exclusion chromatography via an ÄKTA fast protein liquid chromatography system (GE Healthcare). Purity of proteins was determined by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie staining.
4
Purification of MpIspS Enzyme
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The MpIspS expressing cells were harvested from culture broth and disrupted on ice using ultrasonication (Thermo Fisher Scientific, Waltham, MA, USA) in buffer A (50 mM sodium monophosphate, 300 mM NaCl, 10 mM imidazole, and 0.1 mM phenylmethylsulfonyl fluoride as a protease inhibitor). Unbroken cells and cell debris were removed by centrifugation at 14,000 rpm for 10 min at 4 °C, and the supernatants were filtered through a 0.45 μm filter and applied to an IMAC column (Bio-Rad) equilibrated with buffer A. Supernatants collected from lysates were loaded into the Profinia™ Purification System (Bio-Rad). Supernatants were loaded onto a 1-mL IMAC cartridge and washed twice with 5 and 10 mM imidazole buffer A. Proteins were eluted with 250 mM imidazole in buffer A. Imidazole and other salts were removed and changed with 50 mM MOPS buffer (pH 6.0) using a desalting cartridge. The resulting solution was used as the purified MpIspS enzyme. The protein concentration was quantified by the standard Bradford method [44 (link)]. The purified proteins were confirmed by SDS-PAGE.
5
Purification and Characterization of Egg Yolk IgY
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Approximately 385 nmol of Y4‐4 peptide was immobilized on a HiTrap Streptavidin HP column (1 mL; GE Healthcare), according to the manufacturer's instructions. Egg yolk IgY was extracted from a commercial egg by injecting distilled water directly into the peptide‐immobilized affinity column (1 mL), which was connected to a Profinia purification system (Bio‐Rad). Washing buffer (PBS) was used to remove unbound materials. Binding IgY was eluted with 0.1 M glycine‐HCl/0.25 M NaCl (pH 2.5 and pH 3), and the eluate was immediately neutralized with neutralization buffer (1 M Tris–HCl, pH 8.5) and stored at 4 °C until use. The obtained fractions were evaluated for immunoreactivity by enzyme‐linked immunosorbent assay (ELISA) and purity by SDS‐PAGE analysis. From the SDS‐PAGE data, the purity of the obtained fractions was estimated using GelAnalyzer2010a software. Protein concentrations were estimated from the absorbance at 280 nm using an absorbance coefficient of 1.55 mL/mg for IgY and other proteins.
6
Immunopeptidomics of INS-1E Cells
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The unchallenged INS-1E and their derived GRP94 KO and control KO (cells targeted with lentiviral particles still expressing GRP94 similar to unchallenged INS-1E) cells were cultured and maintained between passage numbers 54–72 in RPMI-1640 as described previously [17 (link)]. To perform immunopeptidomics, cells from confluent flasks were detached using EDTA (10 mM), centrifuged at 4000 rpm to pellet the cells. The cell pellets of 5 × 108 per replicate per group were snap-frozen in liquid nitrogen and stored at −80 °C until later use for immune-affinity capture of RT1.A.
The hybridoma cell line OX-18 was cultured in roller bottles in RF5 medium (RPMI-1640 supplemented with 5% fetal bovine serum (FBS), 1% Penicillin-Streptomycin, 2 mM MEM non-essential amino acids, 100 mM HEPES, 2 mM L-glutamine and 50 µM β-mercaptoethanol). The supernatants were collected and antibody harvested by protein G chromatography using a Profinia purification system (Bio-rad®, South Granville, Australia).
The hybridoma cell line OX-18 was cultured in roller bottles in RF5 medium (RPMI-1640 supplemented with 5% fetal bovine serum (FBS), 1% Penicillin-Streptomycin, 2 mM MEM non-essential amino acids, 100 mM HEPES, 2 mM L-glutamine and 50 µM β-mercaptoethanol). The supernatants were collected and antibody harvested by protein G chromatography using a Profinia purification system (Bio-rad®, South Granville, Australia).
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