Protein A Mag Sepharose® Xtra and Protein G Mag Sepharose® Xtra products are magnetic beads designed for high capacity small-scale puriﬁcation/screening of monoclonal and polyclonal antibodies from various species.
Protein A Mag Sepharose® Xtra and Protein G Mag Sepharose® Xtra provide ﬂexible puriﬁcation allowing a wide range of sample volumes and easy scaling up by varying the bead quantity.
The magnetic bead format has excellent properties for small-scale experiments. The high density of the beads allows rapid capture by magnetic devices while the visibility of the beads ensures reliable collection of the bound antibodies in the puriﬁcation procedure. The products are provided with protocols optimized for antibody puriﬁcation.
MagRack™ 6 enables preparation of up to six samples captured in 1.5 mL microcentrifuge tubes (Figure 3.27). When the tubes are placed in the rack, the magnetic beads are attracted to the magnet within a few seconds. This allows easy removal of the supernatant whereas the magnetic beads are left in the tube. MagRack™ Maxi is suitable for volumes up to 50 mL, for example, capture of antibodies in low titer from a larger volume.
Figure 3.27.The high density of Mag Sepharose® beads allows rapid capture by MagRack™ 6 magnetic device.
For immunoprecipitation, it is recommended to use the Protein A Mag Sepharose® and Protein G Mag Sepharose. These products have optimized capacities for immunoprecipitation applications, see Immunoprecipitation techniques for more information.
Protein A Mag Sepharose® Xtra and Protein G Mag Sepharose® Xtra are intended for single use only.
1.5 mL Eppendorf tubes and MagRack™ 6 should be used in the protocol. When using volumes above 1.5 mL, for example larger volumes up to 50 mL, MagRack™ maxi is recommended.
General magnetic separation step
Dispensing the medium slurry
Handling of liquid
Refer to Desalting and buffer exchange for general considerations.
Check the pH of the sample, and adjust if necessary before applying the sample to the beads. The pH of the sample should equal the pH of the binding buffer. Adjusting the pH could be done by either diluting the sample with binding buffer or by buffer exchange using PD MiniTrap™ G-25 or HiTrap™ Desalting.
Clariﬁcation of sample might be needed before applying it to the beads.
Binding buffer: PBS (137 mM sodium chloride, 2.7 mM potassium choride, 100 mM sodium hydrogen phosphate, 2 mM potassium hydrogen phosphate), pH 7.4
Elution buffer: 100 mM glycine-HCl, pH 2.8
Neutralizing buffer: 1 M Tris-HCl, pH 9.0
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µm ﬁlter before use.
This protocol is suitable for most antibody puriﬁcations.
Magnetic bead preparation
Washing (perform this step two times totally)
As a safety measure to preserve the activity of acid-labile antibodies, we recommend the addition of 1 M Tris-HCl, pH 9.0 to tubes used for collecting antibody-containing fractions.
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