ANALYTICAL AND LABORATORY EQUIPMENT 9
Benefits of small-scale antibody purification using magnetic beads
A well-known method for purification of antibodies from serum, ascites, and cell culture supernatant is to utilise the strong affinity of the Fc part of IgG to protein A from Staphylococcus or protein G from Streptococcus.
Une méthode réputée de purification des anticorps à partir de sérum, d’ascite et de surnageant de culture cellulaire consiste à utiliser l’affinité puissante de la partie Fc des IgG à la protéine A des staphylocoques ou à la protéine G des streptocoques.
Eine bekannte Methode zur Reinigung von Antikörpern aus Serum, Ehrlich-Ascites-Tumoren und Zellkultur-Supernatant ist die Ausnutzung der starken Ähnlichkeit des Fc-Teils von IgG gegenüber dem Protein A von Staphylokokken oder Protein B von Streptokokken.
P
urification of antibodies is carried out to concentrate and enrich antigen-
specific antibodies and reduce the background by removing non- specific proteins.
A well known method for purifying antibodies from serum, ascites, and cell culture supernatant is to utilize the strong natural binding affinity of the Fc part of IgG to protein A from Staphylococcus or protein G from Streptococcus. Te binding strengths of protein A and protein G for immunoglobulins depend on the source species and the subclass of the particular immunoglobulin.
For small scale purification and screening of antibodies, in the µl to ml range, there is now the option to use commercially available magnetic separation techniques. Tese procedures utilise protein A and protein G affinity ligands immobilised on magnetic particles/
beads and have several advantages including easy handling of samples and parallel antibody purification. Additional the equipment to prepare the samples using magnetic beads is a simple to use magnetic device that gives low investment cost.
Method - simplicity and speed GE Healthcare has developed Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra magnetic beads, which are designed for efficient, high capacity small-scale purification/screening of monoclonal and polyclonal antibodies from various species. Antibody purification is carried out in a single tube, where the samples are incubated with the magnetic beads equilibrated with binding buffer. Tis enables the antibodies to be captured by the high specificity protein A or protein G ligands on Mag Sepharose resin. Te tube is then placed in a magnetic rack, where the beads are attracted to the magnet within seconds. Tis allows easy removal of the supernatant whereas the magnetic beads remain in the tube, ready for further processing such as wash out unbound sample and elution of the antibody. Te entire purification procedure can take place in less than 40 minutes.
Te magnetic property of the beads means that they respond to the magnetic field but do not retain magnetic properties when the field is removed (paramagnetic). Tis ability
to become magnetized permits efficient extraction. Te bead format also has other excellent properties for small scale experiments including high density, which allows rapid capture by magnetic devices while the visibility of the beads ensures reliable collection of the antibodies in the purification procedure. Tis separation technique provides flexible purification allowing a wide range of sample volumes and easy scaling up by adjusting the bead quantity. Additionally a large number of samples can also be screened in parallel with high throughput on a robotic device.
Antibody binding capacity In addition to Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra several different magnetic beads for small scale antibody purification are available commercially. Tese include: PureProteome Protein A and PureProteome Protein G from Millipore, BioMag Protein A and BioMag Protein G from Qiagen and Dynabeads Protein A and Dynabeads Protein G from Invitrogen.
An internal benchmark study was carried out, in GE Healthcare laboratories, to investigate the relative binding capacity for antibodies using these different magnetic beads. Te capacity for each one was determined by purifying an excess of human IgG and rabbit IgG according to the relevant manufacturer’s instructions. Comparison of this internally generated data showed that both Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra had considerably higher binding capacities for human IgG and rabbit IgG compared to the other corresponding products. Approximately 380µg of purified human IgG and 780µg of rabbit IgG were obtained in a single run with both Protein A Mag Sepharose Xtra and Protein G Mag Sepharose Xtra.
Fig. 1. (Right) Cartoon of antibody purification protocol using magnetic beads.
Tese amounts were approximately double those achieved with the
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