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.
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.
Important Note: This procedure is not used for the preparation of C18 beads
- In the hood, clean glass syringes 3X with chloroform.
- Measure desired lipids into a glass container. A total of ~4μmol total lipid is required.
- Fill stock tubes with N2 or CO2 before re-sealing. Parafilm to keep O2 out.
- Dry lipids using a stream of inert gas, holding tube at a 45o angle to keep dired lipids at the bottom of the tube.
- Place tubes under a slow flow of inert gas for another 2+ hrs.
- During step 5, clean glass syringes 5-10X with clean chloroform. DO NOT PUT SYRINGES AWAY UNTIL CLEANED.
- 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
- Measure out 2mg of the desired bead.
- Wash 3X in 1ml ddH2O.
- Pellet beads, remove as much water as is possible
- Suspend beads in the liposome preparation prepared previously. Circumvolve at room temperature for 20 min to allow bylayers to self-assemble.
- Pellet beads with a gently centrifugation and remove the liquid phase.
- Beads can be washed 1-2X in PBS or ddH2O if al liposomes must be removed.
- 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
- Measure out 2mg C18 beads. Dilute into 100μl of chloroform.
- Measure out lipids as described in liposome preparation (steps 1-3).
- Before drying chloroform, mix the 100μl of C18 beads with the lipids.
- Dry under inert gas, as described in in lipisome preparation protocol.
- Resuspend beads in 1ml of the desired buffer. Vortex and sonicate to suspend.
- Transfer to 1.5ml tube; beads can be used for upto 1 week if covered in an inert gas.
- Vortex beads 3-4min before each use.
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:
- Rinse beads 5X in ddH2O
- Rinse beads 1X 10min in Triton X-100
- Rinse beads 10X in ddH2O
- Soak 1hr in concentrated hydrochloric acid, ideally with heating over 50oC
- Rinse 4X in ddH2O
- 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.
- Suspend cleaned beads in 100μl of protein-free PBS or ddH2O. To this add 10μl of a 50mg/ml IgG stock solution
- Circumvolve 30-60min at room temperature, or 4oC overnight.
- Wash 1X in PBS.
- Block 1 hour in PBS + 5% BSA.
- Wash 1X in PBS or desired buffer.