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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, in a volume equal to the original bead volume.
B) General binding protocol
1. Add biotinylated molecule
2. Incubate for 30 minutes at room temperature
3. Collect beads by placing the tube or microplate on the magnet for 1-2 minutes
4. Wash beads 3-4 times with washing buffer
5. Resuspend the beads in a suitable buffer and volume for your downstream use.
C) Immunoprecipitation
1. Combine the antigen sample with 10 ?g of biotinylated antibody. 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.
2. Resuspend beads by shaking/vortexing
3. Add 25-50 μL of MagSi-STA beads into a 1.5 mL microcentrifuge tube.
4. Prepare the beads for binding by washing with binding buffer as described in a). Finally resuspend in 500 μL binding buffer.
5. Add the antigen sample/biotinylated antibody 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/wash Buffer to the tube and gently mix. Collect the beads and then discard the supernatant. Repeat this step twice.
8. Add 100 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 100 μL of SDS-PAGE reducing sample buffer to the tube and heat 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 from the antibody, pre-rinse the beads with 300 μL 0.1% Tween-20 in water before adding low pH elution buffer.
** If SDS-PAGE buffer is selected for elution, the eluate will contain streptavidin monomers and dimers and biotinylated antibody along with target antigen.
D) Immunoassays
For direct capture, add MagSi-STA beads to a sample containing biotinylated antibodies. During a short incubation, the biotinylated molecules will bind to the beads. Collect the beads on a magnet and discard the supernatant. The beads are now ready to bind the antigen (analyte) from your sample.
For indirect capture, mix the biotinylated antibodies with your sample containing antigen before addng to the beads. Indirect capture can be advantageous when binding conditions are slow or specific molecule orientation is needed.
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