Protein-A sepharose beads are added to a High Five cell supernatant containing a recombinant protein-IgG chimera.
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Protein A binds the Fc region of immunoglobulins through interaction with the heavy chains. Beads coated with protein A can therefore be used to isolate chimeric recombinant proteins that have been fused to the Fc portion of an antibody. In our lab we have used such chimeric proteins to investigate the avidity and specificity of killer-cell immunoglobulin-like receptors (KIR). The fusion of the extracellular domain of such receptors to the Fc region of an antibody renders these normally membrane bound receptors soluble such that they can be employed in cell-free binding assays.
Isolation and purification of recombinant proteins containing the Fc region of IgG.
Our lab uses an insect cell system to generate recombinant chimeric proteins that fuse the extracellular region of a KIR molecule to the Fc region of an antibody. High-Five cell cultures are transfected with high-titer baculoviral supernatants, incubated for 60 hours and the supernatant harvested by centrifugation.
High-Five supernatant is decanted and filtered through a 0.2uM filter. Protein A –sepharose beads are added to the resultant supernatant (0.5ml per 500ml supernatant) and incubated, rotating at 4°C overnight. The supernatant is decanted, the beads washed with PBS (x3 volume of supernatant) and added to PD-10 column with fret filter. Protein is eluted from the protein-A beads using 0.2M glycine, pH 2.7 and immediately neutralized in 1M Tris, pH 8.
The protein-A beads should be thoroughly mixed prior to use as they clump when stored for any length of time. Many proteins will not be stable in the glycine/Tris mixture. We typically further purify them with a G-25 sephadex column using PBS to elute the protein from the column.
We have found that protein-A beads bind highly specifically and strongly to recombinant proteins containing the Fc region of an IgG molecule. Gel analysis of the proteins eluted using this method shows that there are very few contaminant proteins. Additionally, when used in a binding assay against beads coated with single HLA class I antigens, these proteins show very little background binding, again indicating that they are well purified by protein A sepharose beads.
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Product Name: Recombinanat Protein A-Sepharose 4BSupplier Page