Biomedical Engineering Reference
In-Depth Information
surface. modiication. using. phosphonomethyl. iminodiacetic. acid. (PMIDA).. The. PMIDA-
functionalized.nanoparticles.were.then.linked.with.Ni
2+
.for.puriication.of.6x.His-tagged.
proteins,.with.the.phosphonate.group.of.the.PMIDA.ligand.acting.as.a.surface.anchoring.
agent.on.the.magnetite.nanoparticles.and.the.remaining.free.carboxylic.groups.available.
for.binding.with.the.Ni
2+
.ions..The.particles.were.then.used.to.purify.
Entamoeba histolytica
.
malic.enzyme.overexpressed.in.
Escherichia coli
.
In. a. quite. different. approach,. Xu. and. coworkers. coated. iron. oxide. nanoparticles.
(Fe
3
O
4
). with. silica. using. standard. techniques.. Subsequently. poly(2-hydroxyethyl.
methacrylate).(PHEMA).brushes.were.grown.on.the.SiO
2
-MNPs.by.atom.transfer.radical.
polymerization.(ATRP).from.immobilized.initiators,.followed.by.brush.functionalization.
with.nitrilotriacetate-Ni
2+
.(Xu.et.al..2011)..They.expected.that.the.brushes.would.increase.
protein-binding. capacity,. but. that. growth. could. be. controlled. in. a. manner. that. would.
not.affect. the. magnetic. properties.. They. found. that. the. typical. thickness. of. the. brushes.
was.∼50.nm. and. that. multilayer. binding. of. tagged. protein. led. to. a. binding. capacity. of.
220-245.mg. protein/g. of. beads. with. rapid. (∼5.min). binding.. Equally. importantly,. they.
were.able.to.collect.the.beads.with.a.permanent.magnet.
Tong. et. al.. (2001). developed. a. novel. magnetic. afinity. support. (MAS). for. protein.
puriication.using.an.oxidation-precipitation.method.to.entrap.MNPs.of.Fe
3
O
4
.in.polyvinyl.
alcohol. (PVA).. They. then. generated. micrometer-sized. (∼10.μm). magnetic. particles. by.
cross-linking. the. magnetic. luids. with. glutaraldehyde,. followed. by. modiication. of. the.
particles.with.Cibacron.blue.3GA..These.particles.were.then.used.to.bind.lysozyme..The.
capacity.for.lysozyme.adsorption.was.more.than.70.mg/g.MAS.(wet.weight).at.a.relatively.
low. CB. coupling. density. (3-5.μmol/g).. The. lysozyme. could. be. released. by. treating. the.
bound.particles.with.1.0.M.NaCl..In.a.different.afinity.approach,.Nishio.et.al..synthesized.
MNPs. ∼180.nm. in. diameter. consisting. of. 40.nm. magnetite. particles. coated. with.
poly(styrene-
co
-glycidyl. methacrylate. (GMA))/polyGMA,. by. admicellar. polymerization.
(Nishio. et. al.. 2008).. They. found. that. the. particles. were. very. uniform. (184. ±. 9.nm). and.
stable.in.a.wide.range.of.organic.solvents..They.then.functionalized.these.particles.with.
methotrexate.(MTX).using.ethylene.glycol.diglycidyl.ether.(EGDE).as.a.linker..The.MTX-
functionalized.particles.were.subsequently.used.to.purify.dihydrofolate.reductase.(DHFR).
from.cytoplasmic.fractions.of.HeLa.cells.and.THP-1.cells.
In.a.novel,.rather.unusual.application.of.magnetic.nanoparticles,.Zhang.et.al..(2011).cre-
ated.magenetite.(Fe
3
O
4
).particles.coated.with.cationic.silica.for.isolation.of.plasma.mem-
brane.proteins..Based.on.SEM.and.TEM.characterization,.these.cationic.silica-magnetite.
nanocomposites.(CSMN,.
Fe O SiO NH
3
@ -
+
.).were.typically.200.nm.in.diameter.with.an.
iron.oxide.core..They.then.applied.the.CSMN.to.HuH-7.human.hepatocyte.derived.cel-
lular.carcinoma.cells.to.bind.the.plasma.membrane..The.cells.were.then.lysed.using.a.
combination. of. shear. and. hypotonic. buffer. and. the. plasma. membrane. sheets,. which.
were.coated.with.the.CSMN,.were.puriied.magnetically..Western.blotting.veriied.>10-
fold. enrichment. of. plasma. membrane. proteins. compared. with. whole. cell. lysates.. The.
plasma. membrane. isolates. were. then. separated. by. SDS-PAGE. and. individual. bands.
were.digested.with.trypsin.and.separated.by.C
18
.reversed-phase.hydrophobic.interaction.
chromatography.followed.by.identiication.using.Q-TOF-MS..Of.the.proteins.identiied.
by. this. technique,. 55%. of. those. whose. cellular. localization. could. be. determined. were.
plasma. membrane. proteins. (compared. with. 21%. from. a. whole. cell. homogenate).. An.
additional.∼20%.were.localized.to.various.organelles.although.they.hypothesized.that.
many.of.those.may.have.multiple.localization.sites.so.they.may.not.truly.be.contaminat-
ing.proteins..They.also.noted.that.the.use.of.ammonia.
NH
+
4
2
3
(
)
.rather.than.metal.ions.to.
