Preparation of Silica-Magnetic Beads

Silica (SiO₂) beads and silica-coated magnetic beads are a substrate for the attachments of a broad range of biomolecules through polar interactions.  Lipids, proteins and nucleic acids can all be non-covalently attached to these beads.  Silica beads are widely available; silica-Fe beads are also available from a range of sources.  However, Bioclone provides Fe-SiO₂ beads of a size appropriate for phagocytosis assays.

Lipid-Coated Beads

Note: Both silica beads and C18 beads (nucleosil beads) can be used to prepare lipid-coated beads.  Silica will produce a bi-layer; C18 beads will produce a monolayer.

Liposome Preparation

Important Note: This procedure is not used for the preparation of C18 beads

1. In the hood, clean glass syringes 3X with chloroform.
   
2. Measure desired lipids into a glass container. A total of ~4μmol total lipid is required.
   
3. Fill stock tubes with N₂ or CO₂ before re-sealing.  Parafilm to keep O₂ out.
   
4. Dry lipids using a stream of inert gas, holding tube at a 45^o angle to keep dired lipids at the bottom of the tube.
5. Place tubes under a slow flow of inert gas for another 2+ hrs.
   
6. During step 5, clean glass syringes 5-10X with clean chloroform.  DO NOT PUT SYRINGES AWAY UNTIL CLEANED.
   
7. Suspend lipids in 400μl of PBS or other appropriate buffer.  Sonicate to suspend completely
   

NOTE: Steps 1-3 MUST be done in glass; plastic will bind lipids that are not in liposomes
NOTE: If covered in inert gas, these liposomes will be stable for ~1 week.

Silica Bead Preparation

1. Measure out 2mg of the desired bead.
   
2. Wash 3X in 1ml ddH₂O.
   
3. Pellet beads, remove as much water as is possible
   
4. Suspend beads in the liposome preparation prepared previously.  Circumvolve at room temperature for 20 min to allow bylayers to self-assemble.
   
5. Pellet beads with a gently centrifugation and remove the liquid phase.
6. Beads can be washed 1-2X in PBS or ddH₂O if al liposomes must be removed. 
7. Suspend beads in 200-400μl of the desired buffer.

Note: Binding to beads can be improved, if necessary, by washing 1X in methanol followed by a 5min soak in 5M KOH, followed by 3 washes in ddH2O.  This is be done between steps 1 & 2.

C18 Bead Preparation

1. Measure out 2mg C18 beads.  Dilute into 100μl of chloroform.
   
2. Measure out lipids as described in liposome preparation (steps 1-3).
   
3. Before drying chloroform, mix the 100μl of C18 beads with the lipids.
   
4. Dry under inert gas, as described in in lipisome preparation protocol.
5. Resuspend beads in 1ml of the desired buffer.  Vortex and sonicate to suspend.
6. Transfer to 1.5ml tube; beads can be used for upto 1 week if covered in an inert gas.
7. Vortex beads 3-4min before each use.

Protein-Coated Beads

Proteins do not normally bind effectivly to silica beads.  However, a simple treatment will greatly enhance protein binding.  This protocol is optimized for IgG coating, but should work for most proteins.  Note: steps 1-3 are only required for beads purchased dry.  Beads purchased in a collodial solution can be entered directly into step 4, after a single wash in ddH₂O.

To 2mg of beads:

1. Rinse beads 5X in ddH₂O
   
2. Rinse beads 1X 10min in Triton X-100
   
3. Rinse beads 10X in ddH₂O
   
4. Soak 1hr in concentrated hydrochloric acid, ideally with heating over 50^oC
   
5. Rinse 4X in ddH₂O
   
6. Optional: soak in 1M NaOH for 1 hr, followed by 4 washes.  This step is optional and is only required for proteins which do not coat well.
7. Suspend cleaned beads in 100μl of protein-free PBS or ddH₂O.  To this add 10μl of a 50mg/ml IgG stock solution
   
8. Circumvolve 30-60min at room temperature, or 4^oC overnight.
9. Wash 1X in PBS.
10. Block 1 hour in PBS + 5% BSA.
11. Wash 1X in PBS or desired buffer.