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Protocol
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MagSi-protein G and MagSi-protein A are useful for many applications, including immunoprecipitation, antibody screening, protein interaction studies, phage display, immunoassays, purification of proteins and peptides or nucleic acids, and cell isolation.
Protein A and Protein G bind to the Fc region of immunoglobulins, but have different specificities (Table). The albumin, cell wall and cell membrane binding domains, present on the wild-type protein G, have been removed from the recombinant protein G (26kDa) which is used for MagSi-protein G beads. The 42kDa Staphylococcal Protein A on MagSi-protein A is produced in E. Coli. Immunoglobulins optimally bind to Protein A and Protein G at pH 8, and can be released at low pH (2.5-3) or in protein denaturing conditions.
MagSi-protein A and MagSi-protein G beads are offered in sizes of 600 nm and 1.0 μm. The sedimentation time of 600nm MagSi beads has been optimized and is approx. 4 times longer compared to 1.0 μm beads. This allows e.g. long incubation times without shaking/mixing etc. MagSi beads with a diameter of 1μm have stronger magnetic properties and will separate approx. 2x faster compared to 600nm in the same conditions, the typical separation time is ≥ 1 minute using a suitable magnet.
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Assay Procedure
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A) Bead preparation procedure
1. Resuspend beads by shaking/vortexing
2. Pipette the required volume of beads into a tube or microplate (10-20 μL is suitable as a starting point)
3. Collect beads by placing the tube or microplate on the magnet for 1-2 minutes
4. While tube/micro plate is still on the magnet, carefully remove supernatant without touching the bead pellet
5. Take tube/micro plate from the magnet and add Washing Buffer
6. Resuspend beads by vortexing or pipetting
7. Repeat step 3 - 5 at least 3 times
8. Finally resuspend the beads in a suitable buffer for your downstream use, in a volume equal to the original volume.
B) Immunoprecipitation
1. Resuspend beads by shaking/vortexing
2. Add 25-50 μL of MagSi-protein A or MagSi-protein G beads into a 1.5 mL microcentrifuge tube.
3. Prepare the beads for binding by washing with Binding Buffer as described in a). Finally resuspend in 500 μL Binding Buffer.
4. Combine the antigen sample with 2-4 ?g of IgG. Dilute each sample to a minimum volume of 300 μL with cell lysis buffer or Binding/Wash Buffer. Incubate 1-2 hours at room temperature or overnight at 4 °C with mixing.
5. Add the sample mixture to the 1.5 ml microcentrifuge tube containing pre-washed magnetic beads (3.) and incubate at RT for 30 minutes with mixing.
6. Collect beads by placing the tube on the magnet for 1-2 minutes, pipette off and save the supernatant for analysis.
7. Add 300 μL of Binding/Washing Buffer to the tube and gently mix. Collect the beads and then discard the supernatant. Repeat this step twice.
8. Add 50 μL of Elution Buffer to the tube. For low pH elution, incubate the tube at room temperature with mixing for 5 minutes. For SDS-PAGE elution, add 50 μL of SDS-PAGE reducing sample buffer to the tube and incubate the samples at 90 °C for 10 minutes.
9. Collect beads by placing the tube on the magnet for 1-2 minutes and transfer the supernatant containing target antigen.
*Low pH elution buffers are effective for most antibody-antigen interactions, however, to ensure efficient release of target antigen, pre-rinse the beads with 300 μL 0.1% Tween-20 in water without buffer before adding low pH elution buffer.
** If co-elution of the target antigen and the antibody is unfavourable in the application the antibody should be crosslinked. Protocols for crosslinking are available from MagnaMedics under request.
C) Antibody purification
1. Resuspend beads by shaking/vortexing
2. Add 100 μL of MagSi-protein A or MagSi-protein G beads into a 1.5 mL microcentrifuge tube.
3. Prepare the beads for binding by washing with Binding Buffer as described in a). Finally resuspend in 90 μL binding buffer.
4. Add 10 μL serum sample. Incubate 1-2 hours at room temperature or overnight at 4 °C with mixing.
5. Collect beads by placing the tube on the magnet for 1-2 minutes, pipette off and save the supernatant for analysis.
6. Add 100 μL of Binding/Washing Buffer to the tube and gently mix. Collect the beads and then discard the supernatant. Repeat this step twice.
7. Add 50 μL of Low pH elution buffer to the tube. Incubate the tube at room temperature with mixing for 5 minutes.
8. Collect beads on the magnet for 1-2 minutes and transfer the supernatant containing purified antibodies.
Practical Notes:
Optimize the quantity of beads for each application
In case the volume of sample for C) is more than 25 % of the bead volume, equilibrate the sample first with a 0.5 M Phosphate buffer pH 8 5X stock solution (final molarity 0.1 M).
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