Considerations for coupling
Ligand use
MagSi-Direct allows optimal coupling at low concentrations of antibody or protein (10 μg/mg beads). Low affinity ligands may require increasing the amount of input.
The optimal coupling range is ligand dependant and determined empirically. Using excess ligand significantly increases consumption. If it is not a cost factor, it is recommended to use u to 50 μg per mg beads. However, an excess of the ligand will increase the potential for leakage in downstream use.
The presence of antibody aggregates also increases the potential for leakage. This effect can be reduced by removing the aggregates by centrifugation at 16,000 x g for 10 minutes at 4 °C
Different biological molecules such as antibodies, proteins, enzymes, etc. have different characteristics. Even antibodies raised in the same species against the same antigen can vary greatly. As a consequence, the coupling efficiency may vary between different batches. The stability is also dependent on ligand used.
In order to optimize the coupling of antibodies/proteins, the user should consider the buffer composition, preservatives and stabilizing proteins used to store the antibodies/proteins.
Buffer composition
In case the storage buffer of the biological molecules contain amine groups within their ingredients (e.g. Tris), the buffer may need to be exchanged with 1X Immobilization Buffer by dialysis prior to coupling antibodies/proteins to the beads.
Preservatives
Some preservatives included in buffers (sodium azide, thimerosal) may lead to a small decrease in antibody coupling efficiency. For most applications it will not have a significant effect, but if necessary it can be compensated by slightly increasing the quantity of antibody used in the coupling reaction. Alternatively, preservatives can be removed prior to coupling by standard gel filtration chromatography or dialysis against 1X Immobilization Buffer.
Stabilizing reagents
Storage buffers of antibodies, proteins and enzymes may contain protein additives such as BSA or gelatin. If untreated, these additives may be coupled to the bead surface along with the antibody or protein.
Glycerol
Some proteins (enzymes) are supplied in glycerol. Coupling of antibodies/proteins stabilized in glycerol is not recommended. Although it is possible, the functionality may be negatively effected. Glycerol can be exchanged with 1X Immobilization Buffer by dialysis prior to coupling.

Prepare a 1X Immobilization Buffer:
Dilute the 10X Immobilization Buffer (0.25 M MES pH 5.5 Proclin 300 0.5%) stock solution 10x in ddH2O

The Blocking Buffer included in the kit contains BSA as a blocking agent. If the user requires a different blocking agent (gelatin, HSA, casein, etc.), prepare a Blocking Buffer containing the blocking agent of choice in 1X Immobilization Buffer (the standard Blocking Buffer is the same as 1X Immobilization Buffer, but includes 0.1% BSA)

Aliquot and wash the beads
- 1. Vortex MagSi-Direct Particle Mix to fully resuspend the beads.
- 2. Aliquot 1 mL of MagSi-Direct Particle Mix to a 1.5 mL tube.
- 3. Place the tube on the magnetic separator for 2 minutes to collect the magnetic beads and discard the supernatant.
- 4. Wash the beads twice with 1 mL of ddH2O (vortex at high speed for 10 seconds, collect the beads and discard the supernatant as above).
- 5. Add 500 μL ddH2O and vortex at for 10 seconds.

Activate the beads with Shake&Run Coating Solution
- 6. Add 500 μL Shake&Run Coating Solution to a fresh 1.5 mL tube. While vortexing at moderate speed, slowly add the 500 μL of beads from step 5. Continue vortexing for 10 seconds.
- 7. Incubate for 60 minutes at RT. Use a rotating mixer to ensure that the beads are kept in suspension.
- 8. Collect the magnetic beads using the magnetic separator and discard the supernatant.
Optional:
Wash the beads twice with 1 mL of ddH2O as in step 4 and store the Shake&Run activated beads at 2-8°C (do not freeze), or directly go to step 9.

Couple your protein to the beads
- 9. Wash the pelleted beads from step 8 twice with 1 mL 1X Immobilization Buffer. Vortex, separate andn discard the supernatant each time. Resuspend the beads in 250 μL 1X Immobilization Buffer and vortex thoroughly to resuspend the beads.
- 10. Prepare a solution of the antibody or protein in 1X Immobilization Buffer. Use a final concentration of 400 μg/mL.
- 11. While continuously mixing the protein solution at moderate speed, slowly add the washed bead suspension from step 9. Incubate for 60 minutes at room temperature on a rotating mixer.
- 12. Wash twice with 1 mL 1X Immobilization Buffer. Vortex, separate and discard the supernatant each time.
- 13. Add 1 mL of Blocking Buffer and vortex at moderate speed for 10 seconds. Incubate for 60 minutes at room temperature on a rotating mixer.
- 14. Collect the magnetic beads using the magnetic separator and discard the supernatant.
- 15. Wash twice with 1 mL 1X Immobilization Buffer. Vortex, separate and discard the supernatant each time.
- 16. Add the 1 mL of ddH2O, 1X Immobilization Buffer or a different storage buffer of choice depending on the application. Optionally add a detergent. In case the beads are stored, add a preservative.
- 17. Store the beads at 2-8°C until use. Do not freeze.