10 ANALYTICAL AND LABORATORY EQUIPMENT
PureProteome Protein A and G (approximately 230µg of purified human IgG and 550 µg of rabbit IgG) and four-times greater than with the other commercially beads.
in 50ml diluted hybridoma cell supernatant (0.07mg Ab/ ml) was successfully purified and concentrated using 1.75ml Protein A Mag Sepharose Xtra. Te sample
Purification One of the key advantages of magnetic bead purification is the ability to use different volumes of sample and bead slurry. In this study, low expressed monoclonal mouse IgG2b
load was 20 mg/ml sedimented medium and the experiment was performed in duplicate. Te results, illustrated in Fig. 2, showed high specificity according to SDS- PAGE analysis and recoveries of ~70 per cent. Te purified mouse IgG2b
was concentrated from 50 ml to 3.5 ml.
Repeatable antibody purification To show the repeatability of Protein G Mag Sepharose Xtra, six replicate purification runs were performed. Te load was half of the total binding capacity of the medium.
Te yield and purity of the eluted fractions were determined via absorbance measurements and SDS-PAGE analysis respectively. High repeatability, as shown in Fig. 3, was demonstrated for both yield and purity using Protein G Mag Sepharose Xtra (RSD <2 per cent).
In summary, protein A or protein G magnetic beads for antibody purification provides a simple, easily-handled tool for rapid separations that is scalable across the µl to ml range. A benchmark study demonstrated that GE Healthcare’s
Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra had considerably higher binding capacities for both human IgG and rabbit IgG than other commercially available products.
Furthermore, experiments demonstrated that antibodies can be reproducibly purified with high recovery and purity.
Ann Bergh, Fredrik Borg, Stefan D Eriksson, Gunnar Glad, Therese Granér, Helena
Hedlund and Ulf Hellberg, GE Healthcare Bio-Sciences AB, Uppsala, Sweden.
www.gelifesciences.com
Study parameters: Medium:
Sample Volume: Binding Buffer: Elution Buffer:
Lanes 1. Molecular weight markers
2. Sample 3. Eluted pool, purification 1 4. Eluted pool, purification 2
Protein A Mag Sepharose Xtra
Bead Slurry Volume: 1.75 ml (175 µl sedimented medium) Sample:
Mouse IgG2b from hybridoma cells
50 ml (25 ml cell supernatant diluted with 25 ml binding buffer)
PBS (10 mM phosphate, 140 mM NaCl, 2.7 mM KCl), pH 7.4 100 mM glycine-HCl, pH 2.8
Fig. 2. (Above) SDS-PAGE analysis (reduced conditions, Deep Purple total protein stain) of the purification of mouse IgG2b
using Protein A Mag Sepharose Xtra.
Fig. 3. (Below) Six replicate purification runs on Protein G Mag Sepharose Xtra. Left: Recovery of purified protein in each purification run. Right: SDS-PAGE analysis (reduced conditions, Deep Purple total protein stain).
Study parameters: Medium:
Sample Volume: Binding Buffer:
Elution Buffer:
www.scientistlive.com
Protein G Mag Sepharose Xtra
Bead Slurry Volume: 100 µl (10µl sedimented medium) Sample:
Human IgG spiked in
E.Coli lysate 300µl
PBS (10 mM phosphate, 140 mM NaCl, 2.7 mM KCl), pH 7.4 100 mM glycine-HCl, pH 2.8
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