Protein was reduced at a concentration of 0.4 mg/ml in 2 M arginine, 20 mM sodium phosphate (NaPO4, 0.1 M NaCl), 40 mM EDTA at pH 8.0 with 0.1 M DTT for 1 h at 20°C. This process was followed by removal of the reductant and buffer exchange on a PD-10 column to an identical buffer with 25 mM arginine rather than 2 M. PEGylation reagent was added (1:0.8 molar ratio) and incubated for 4 h at 4°C. This solution was then buffer exchanged to 20 mM sodium acetate with 0.05% Tween at pH 6.0 for cation exchange purification on a 5 ml SP-HP column (GE Healthcare) with a linear gradient from 20% buffer containing 1 M NaCl to 100% of the same buffer at 2 ml/min for 1 h. Fractions containing protein of the expected size of PEG-DI were identified by peaks on a chromatogram at 280 nm and then pooled. The hexahistidine tag was cleaved using FXa as in McDonnell et al. (15 (link)). Quantification from this point onwards was by BCA for both PEGylated and non-PEGylated form, thus 20 μg of WT-DI contains the same amount of DI as 20 μg WT-PEG-DI.
Sp hp column
Manufactured by GE Healthcare
The SP HP column is a size exclusion chromatography column designed for the purification and analysis of proteins and other biomolecules. The column is constructed with a resin that allows for the separation of molecules based on their size and shape. Its core function is to facilitate the separation and purification of target biomolecules from complex mixtures.
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Lab products found in correlation
Protein g affinity column, by GE Healthcare (2 mentions)
Hek293 6e cells, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 expression, by Thermo Fisher Scientific (2 mentions)
0.2 mm filter unit, by Sartorius (2 mentions)
G25 desalting column, by GE Healthcare (2 mentions)
Complete edta free protease inhibitor, by Merck Group (1 mentions)
Superdex 75 size exclusion column, by GE Healthcare (1 mentions)
Superdex 75 10 300 gl, by GE Healthcare (1 mentions)
7 protocols using sp hp column
1
PEGylation and Purification of DI Protein
2
Isolating Fab-PEG Conjugates Using SPHP Column
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A 5 mL prepacked SPHP column (GE Healthcare) was used to further isolate the desired product. The conjugate pool was titrated down to pH 5.0 using acetic acid and loaded onto the column. Buffer A, composed of 20 mM sodium acetate, pH 5.0 was used to wash away non-specific contaminants, while buffer B comprised of buffer A with the addition of 1M NaCl was employed for elution. A very shallow gradient from 0–20% buffer B, over 50 CVs was successful in isolating the 8x Fab + 8-arm PEG from the remaining impurities as demonstrated in S2B Fig .
3
Cloning and Purification of Staphylococcal Alpha-Toxin Mutants
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The wild-type hla gene was PCR amplified from S. aureus SF8300 (USA300) genomic DNA and cloned into a pCN-based Escherichia coli -staphylococcal shuttle vector under the control of a constitutive promoter based on the S. aureus clpB gene promoter (36 (link), 37 (link)). Alanine mutant hla expression plasmids were prepared by cloning synthetic DNA fragments containing the mutations into the wild-type expression construct. The alanine mutant plasmids were introduced into the S. aureus RN4220Δhla strain by electroporation and selected on medium containing 10 μg/ml chloramphenicol. Mutant-expressing strains were cultured overnight at 37°C in BHI (brain heart infusion) broth (Criterion, Inc.) with 10 μg/ml chloramphenicol, and AT proteins were purified from culture supernatants by cation exchange chromatography using an SP-HP column (GE Healthcare) equilibrated with 30 mM Na-acetate, pH 5.2, 20 mM NaCl, 1 mM EDTA and eluted with a linear gradient to 500 mM NaCl.
4
Production and Purification of mAb1 Fab
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The genes of light chain and heavy chain of mAb1 Fab fragment were synthesized with CD33 signal peptide and cloned into the vector pJSV002 for mammalian cell expression. The plasmids were then transfected into HEK293-6E cells (obtained from Invitrogen) at density of 1.0 × 106 cells ml−1 with DNA molar ratio of 1:1. The transfection was performed following Invitrogen’s Freestyle_293 expression manual. The cell culture supernatant was filtered after 120 h post transfection and applied to a protein G affinity column (GE Healthcare) that was pre-equilibrated in phosphate buffered saline (PBS). The bound Fab was eluted with 100 mM glycine-HCl, pH 2.8. Fractions were collected and neutralized immediately with 1/20 volume of 2 M Tris-HCl, pH 9.0. The pooled fraction was then diluted into 20 mM Na acetate, pH 5.5 and applied to a SP HP column (GE Healthcare). The bound Fab was eluted with a 100–300 mM linear gradient of NaCl in 20 mM Na acetate, pH 5.5 and buffer-exchanged to PBS on a G25 desalting column (GE Healthcare). Purified protein was sterilized by filtration through a 0.2 mm filter unit (Sartorius). The purity of the protein sample was analyzed by SDS-PAGE and size-exclusion chromatography. The Fab identity was confirmed by mass spectrometry.
5
Purification of Recombinant Protein from E. coli
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Protein was purified from E. coli. Cells were grown at 37 °C until an optical density (OD) of 0.8 was reached and then induced with 400 μM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Protein was expressed for h hours at 28 °C. Cells were collected in lysis buffer (50 mM TRIS pH 7.5, 150 mM NaCl, 1 mM TCEP, 2 mM Imidazole) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. Lysate was cleared by spinning down at 21,000 × g. Perchloric acid (2%) was slowly added to the supernatant while stirring on ice. The sample was cleared again by centrifugation at 20,000 × g and the supernatant was dialyzed against 50 mM ammonium acetate pH 4.5. The sample was then loaded on a SP HP column (GE Healthcare) and eluted with a linear salt gradient ranging from 0 to 500 mM NaCl. The sample was subsequently purified on a Superdex75 size exclusion column (GE Healthcare) in 50 mM TRIS pH 8.0 and 100 mM NaCl.
6
Purification of His-tagged Proteins from E. coli
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H1.1, H1.2, and all forms of H1.1 variants, including truncated and chimeric H1, were cloned into a pET28a vector and expressed with a C-terminal 6xHis-tag in Escherichia coli strain BL21 (DE3) in the presence of 0.1 mM IPTG overnight at 16 °C. Cells were collected by centrifugation and resuspended in lysis buffer: 20 mM Tris–HCl, 500 mM NaCl, pH 8.0. The cells were then lysed by ultrasonication. After centrifugation at 18,000 × g, the supernatant was applied to a Chelating SFF(Ni) column, and target proteins were eluted with 250 mM Imidazole. The resultant proteins were further purified by cation-exchange chromatography using an SP HP column (GE Healthcare). The eluted peaks were applied to a Superdex 75 10/300GL (GE Healthcare) gel filtration column, then dialyzed and concentrated in a stock buffer: 20 mM HEPES, 150 mM NaCl, pH 8.0.
7
Production and Purification of mAb1 Fab
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