Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

Summary

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

Abstract

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Introduction

Reactive polymer brushes have received much interest in recent years. They can be used to immobilize functional molecules on organic or inorganic materials to create activated surfaces with applications in areas such as detection and separation^1,2,3,4,5. Among the reactive polymers reported, those containing pentafluorophenyl ester units are particularly useful due to their high reactivity with amines and resistance toward hydrolysis⁶. One such polymer is poly(PFPA), and it can be re....

Protocol

1. Preparation of Poly(PFPA) Homopolymer

1. Recrystallization of AIBN
   1. Combine 5 g of 2,2’-azobis(2-methylpropionitrile) (AIBN) with 25 mL of methanol in a 250 mL beaker. Immerse the beaker in a 60 °C oil bath, then vigorously stir the mixture with a stir bar until AIBN is fully dissolved.
   2. Filter the warm solution through filter paper (5-8 μm particle retention) and store the filtrate at 4 °C to allow the crystals to form slowly.
   3. Collect the recrystallized AIBN by filtration. Combine the collected product with 25 mL of fresh methanol and repeat the recrystallization process.
   4. Dry the 2x recrystal....

Results

A schematic for the preparation of poly(PFPA) grafted SiO₂ beads, with or without PEG substitution is shown in Figure 1. To monitor the APTMS and poly(PFPA) grafting process, bare SiO₂ beads, APTMS functionalized SiO₂ beads, and poly(PFPA) grafted SiO₂ beads are characterized by both DLS (Figure 2) and XPS (Figure 3). IP efficiencies of the beads are determ.......

Discussion

The synthesis of poly(PFPA) grafted SiO₂ beads is illustrated in Figure 1. By employing APTMS as a linker molecule, poly(PFPA) brushes covalently grafted to SiO₂ substrate can be prepared via a simple two-step process. Although some of the PFP units are sacrificed for the reaction with APTMS, a large number of the PFP units are expected to remain available for later reaction with either amino-PEG or antibodies. The PFP groups are known to form low energy surfaces so pol.......

Materials

Name	Company	Catalog Number	Comments
2,2-Azobisisobutyronitrile, 99%	Daejung Chemicals	1102-4405
Methyl alcohol for HPLC, 99.9%	Duksan Pure Chemicals	d62
Phenylmagnesium bromide solution 1.0 M in THF	Sigma-Aldrich	331376
Carbon disulfide anhydrous, ≥99%	Sigma-Aldrich	335266
Benzyl bromide, 98%	Sigma-Aldrich	B17905
Petroleum ether, 90%	Samchun Chemicals	P0220
Ethyl ether, 99%	Daejung Chemicals	4025-4404
Magnesium sulfate anhydrous, powder, 99%	Daejung Chemicals	5514-4405
Pentafluorophenyl acrylate	Santa Cruz Biotechnology	sc-264001	contains inhibitor
Aluminium oxide, activated, basic, Brockmann I	Sigma-Aldrich	199443
Sodium Chloride (NaCl)	Daejung Chemicals	7548-4400
Anisole anhydrous, 99.7%	Sigma-Aldrich	296295
Silica nanoparticle	Microparticles GmbH	SiO2-R-0.7	5% w/v aqueous suspension
3-Aminopropyltrimethoxysilane, >96.0%	Tokyo Chemical Industry	T1255
Dimethyl sulfoxide for HPLC, ≥99.7%	Sigma-Aldrich	34869
Amino-terminated poly(ethylene glycol) methyl ether	Polymer Source	P16082-EGOCH3NH2
Phosphate buffered saline tablet	Takara	T9181
Tween-20	Calbiochem	9480
Tris-HCl (pH 8.0)	Invitrogen	AM9855G
KCl	Invitrogen	AM9640G
NP-40	VWR	E109-50ML
Glycerol	Invitrogen	15514-011
Dithiothreitol	Biosesang	D1037
Protease inhibitor	Merck	535140-1MLCN
Bromo phenol blue	Sigma-Aldrich	B5525-5G
Tris-HCl (pH 6.8)	Biosolution	BT033
Sodium dodecyl sulfate	Biosolution	BS003
2-Mercaptoethanol	Gibco	21985-023
PKR Antibody	Cell Signaling Technology	12297S
GAPDH Antibody	Santa Cruz Biotechnology	sc-32233
Normal Rabbit IgG	Cell Signaling Technology	2729S
HeLa	Korea Cell Line Bank	10002
Sonicator	DAIHAN Scientific	WUC-D10H
Ultrasonicator	BMBio	BR2006A
Centrifuge I	Eppendorf	5424 R
Centrifuge II	LABOGENE	1736R
Rotator	FINEPCR	ROTATOR/AG
Vacuum oven	DAIHAN Scientific	ThermoStable OV-30
Gel permeation chromatography (THF)	Agilent Technologies	1260 Infinity II
X-ray photoelectron spectrometer	Thermo VG Scientific	Sigma Probe
Dynamic light scattering	Malvern Instruments	ZEN 3690