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2021-02-01T19:38:33Z

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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, [http://www.bioclone.us/ Bioclone] provides Fe-SiO2 beads of a [http://www.bioclone.us/Silica-modified-magnetic-beads-particle-resin-matrix.html 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 

#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 
<pre>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.
</pre>
===== 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.
<pre>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.
</pre>
===== 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.

 

== 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: 

#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.

Preparation of Silica-Magnetic Beads - Revision history

Admin: Created page with "Silica (SiO2) beads and silica-coated magnetic beads are a substrate for the attachments of a broad range of biomolecules through polar interactions. Lipids,..."

Admin: Created page with "Silica (SiO₂) beads and silica-coated magnetic beads are a substrate for the attachments of a broad range of biomolecules through polar interactions. Lipids,..."