Introduction
Important information before using Sera-Mag SpeedBeads Protein A/G Magnetic Particles
Procedure for manual antibody purification
Procedure for automated antibody purification
Procedure for manual immunoprecipitation (IP)
Procedure for automated immunoprecipitation
Troubleshooting
Materials
Sera-Mag SpeedBeads Protein A/G Magnetic Particles (Table 1) provide a fast and convenient method for both manual and automated magnetic isolation of proteins using affinity binding. The particles can be used for isolating antibodies from serum, cell culture supernatant or ascites, and for immunoprecipitation and co-immunoprecipitation of antigens from cell or tissue extracts. Bound antibodies or antigens are dissociated from the particles using an elution buffer.
The particles can be manually removed from the solution using a magnetic stand, or by automation using automated magnetic particle handling systems.
Sera-Mag SpeedBeads Protein A/G Magnetic Particles contain a recombinant Protein A/G (Mr ~50 500; apparent molecular weight by SDS-PAGE Mr ~40 000 to 45 000) that combines the IgG binding domains of both Protein A and Protein G.
Protein A/G contains four Fc-binding domains from Protein A and two from Protein G, making it a more general and convenient tool for investigating and purifying immunoglobulins. Also, Protein A/G binding to immunoglobulins is not as pH-dependant as Protein A.
Sera-Mag SpeedBeads Protein A/G particles are uniform, colloidally stable, monodispersed, non-porous superparamagnetic spheres made by a proprietary core-shell method.
The core is a carboxylate-modified particle made by free radical emulsion polymerization of styrene and acid monomer. Two layers of magnetite (Fe3O4) are coated onto this core particle, resulting in faster magnetic response times. The surface is chemically modified with a proprietary method to minimize nonspecific binding of proteins. Finally, Protein A/G is covalently bound to the particle surface.
The particles are supplied at 1% solids (10 mg/ml) in 0.05 % sodium azide and are available in 1 ml, 5 ml and 100 ml package sizes.
Additional materials required
Antibody purification from serum, cell culture supernatant, or ascites
Note: To ensure homogeneity, mix the particles thoroughly before use by repeated inversion, gentle vortexing or using a rotating platform.
Additional materials required
Preparation of instrument and plate set-up
Note: The following protocol is designed for general use with the KingFisher Flex or KingFisher 96 Instrument. The protocol can be modified according to customer needs using the Thermo Scientific BindIt™ software provided with the instrument.
Notes
Executing the antibody purification protocol on the KingFisher Flex
Additional materials required
Immunoprecipitation
Note: This protocol is a general guideline for immunoprecipitation and will require optimization for each application.
Additional materials required
Instrument preparation and plate set-up
Note: The following protocol is designed for general use with the KingFisher Flex or KingFisher 96 Instrument. The protocol can be modified according to your needs using the BindIt Software provided with the instrument.
Notes
Executing automated immunoprecipitation protocol
Troubleshooting |
---|
如要继续阅读,请登录或创建帐户。
暂无帐户?