Name: preparation of poly(pentafluorophenyl acrylate) functionalized sio2 beads for protein purification
Uploaded: 2018-11-19T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification at JoVE.com

This work was supported by Agency for Defense Development (Grant No. UD170039ID).

1. Preparation of Poly(PFPA) Homopolymer
<ol>
	<li>Recrystallization of AIBN
	<ol>
		<li>Combine 5 g of 2,2&#8217;-azobis(2-methylpropionitrile) (AIBN) with 25 mL of methanol in a 250 mL beaker. Immerse the beaker in a 60 &#176;C oil bath, then vigorously stir the mixture with a stir bar until AIBN is fully dissolved.</li>
		<li>Filter the warm solution through filter paper (5-8 &#956;m particle retention) and store the filtrate at 4 &#176;C to allow the crystals to form slowly.</li>
		<li>Collect the recrystallized AI...

<ol>
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J., Theato, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+surface+coatings+based+on+polysilsesquioxanes:+controlled+functionalization+for+specific+protein+immobilization.">Reactive surface coatings based on polysilsesquioxanes: controlled functionalization for specific protein immobilization.</a> Langmuir. 25 (17), 10068-10076 (2009).</li><li>Kessler, D., Theato, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+surface+coatings+based+on+polysilsesquioxanes:+defined+adjustment+of+surface+wettability.">Reactive surface coatings based on polysilsesquioxanes: defined adjustment of surface wettability.</a> Langmuir. 25 (24), 14200-14206 (2009).</li><li>Kessler, D., Nilles, K., Theato, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+approach+towards+multi-functional+surfaces+with+adjustable+and+dual-responsive+wettability+using+a+hybrid+polymer+toolbox.">Modular approach towards multi-functional surfaces with adjustable and dual-responsive wettability using a hybrid polymer toolbox.</a> Journal of Materials Chemistry. 19 (43), 8184-8189 (2009).</li><li>Eberhardt, M., Mruk, R., Zentel, R., Theato, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+pentafluorophenyl(meth)acrylate+polymers:+new+precursor+polymers+for+the+synthesis+of+multifunctional+materials.">Synthesis of pentafluorophenyl(meth)acrylate polymers: new precursor polymers for the synthesis of multifunctional materials.</a> European Polymer Journal. 41 (7), 1569-1575 (2005).</li><li>Jochum, F. 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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...

An erratum was issued for: <a href="https://www.jove.com/video/58843/preparation-poly-pentafluorophenyl-acrylate-functionalized-sio2-beads">Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification</a>.&#160; Throughout the article, the term &#34;3-aminopropyltriethoxysilane&#34; has been replaced with &#34;3-aminopropyltrimethoxysilane&#34;, and &#34;APTES&#34; with &#34;APTMS&#34;.

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

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 separation1,2,3,4,5. 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 readily functionalized post-polymerization with molecules containing primary or secondary amines7,8,9,10. In one example, poly(PFPA) brushes were reacted with amino-spiropyrans to create light-responsive surfaces7.
The preparation of poly(PFPA) and its applications have been described in a number of previous publications6,7,8,9,10,11,12,13,14,15,16,17. In particular, Theato and co-workers reported the synthesis of poly(PFPA) brushes via both &#34;grafting to&#34; and &#34;grafting from&#34; methods7,8,10,11,12. In the &#34;grafting to&#34; approach, a poly(methylsilsesquioxane)-poly(pentafluorophenyl acrylate) (poly(MSSQ-PFPA)) hybrid polymer was synthesized8,10,11,12. The poly(MSSQ) component was able to form strong adhesion with a number of different organic and inorganic surfaces, thus allowing the poly(PFPA) component to form a brush layer on the coated material surface. In the &#34;grafting from&#34; approach, surface initiated reversible addition and fragmentation chain transfer (SI-RAFT) polymerization was employed to prepare poly(PFPA) brushes7. In this case, a surface immobilized chain transfer agent (SI-CTA) was first covalently attached to the substrate via silica-silane reaction. The immobilized SI-CTA then participated in the SI-RAFT polymerization of PFPA monomers, generating densely packed poly(PFPA) brushes with stable covalent linkage to the substrate.
By utilizing the poly(PFPA) brushes synthesized via SI-RAFT polymerization, we recently demonstrated the immobilization of antibodies on poly(PFPA) grafted silica particles and their subsequent application in protein purification18. The use of poly(PFPA) brushes for antibody immobilization was found to resolve a number of issues associated with current protein separation through IP. Conventional IP relies on the use of Protein A/G as a linker for antibody immobilization19,20,21. Since the use of Protein A/G allows the antibodies to be attached with a specific orientation, high target antigen recovery efficiency is achieved. However, the use of Protein A/G suffers from non-specific protein interaction as well as the loss of antibodies during protein recovery, both of which contribute to a high level of background noise. To resolve these shortcomings, direct crosslinking of the antibodies to a solid support has been explored22,23,24. The efficiency of such techniques is typically low due to the random orientation of the crosslinked antibodies. For the poly(PFPA) grafted substrate, the immobilization of antibodies is permanent, achieved through exchange reaction between PFP units and amine functionalities on antibodies. Although the antibody orientation is still random, the system benefits from having many reactive PFP sites, controllable by the degree of polymerization. Furthermore, we showed that by partial substitution of PFP units with amino-PEG, surface hydrophilicity can be tuned, further improving the protein recovery efficiency of the system18. Overall, the poly(PFPA) grafted silica particles were shown to be an effective alternative to traditional IP with reasonable efficiency as well as much cleaner background.
In this contribution, we report an alternative method to prepare poly(PFPA) grafted surface for antibody immobilization and IP application. In a simple two-step process, as illustrated in Figure 1, an APTMS linker molecule is first deposited onto the silica surface, then the poly(PFPA) polymer is covalently attached to the linker molecule through the reaction between the PFP units on the polymer and the amine functions on APTMS. This preparation method allows for the permanent crosslinking of poly(PFPA) to a substrate surface, but avoids the many complications associated with SI-CTA synthesis and SI-RAFT polymerization of poly(PFPA) brushes. Partial substitution of the PFP units with amino-PEG can still be performed, allowing fine-tuning of the polymer brush surface properties. We show the poly(PFPA) grafted silica beads thus prepared can be immobilized with antibodies and used for protein enrichment via IP. The detailed bead preparation procedure, antibody immobilization, and IP testing are documented in this article, for readers interested in seeking an alternative to conventional Protein A/G based IP.

<table>
	<tbody>
		<tr>
			<td>2,2-Azobisisobutyronitrile, 99%</td>
			<td>Daejung Chemicals</td>
			<td>1102-4405</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol for HPLC, 99.9%</td>
			<td>Duksan Pure Chemicals</td>
			<td>d62</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmagnesium bromide solution 1.0&#160;M in THF</td>
			<td>Sigma-Aldrich</td>
			<td>331376</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon disulfide anhydrous, &#8805;99%</td>
			<td>Sigma-Aldrich</td>
			<td>335266</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl bromide, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum ether, 90%</td>
			<td>Samchun Chemicals</td>
			<td>P0220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl ether, 99%</td>
			<td>Daejung Chemicals</td>
			<td>4025-4404</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate anhydrous, powder,&#160;99%</td>
			<td>Daejung Chemicals</td>
			<td>5514-4405</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentafluorophenyl acrylate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-264001</td>
			<td>contains inhibitor</td>
		</tr>
		<tr>
			<td>Aluminium oxide, activated, basic, Brockmann I</td>
			<td>Sigma-Aldrich</td>
			<td>199443</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Daejung Chemicals</td>
			<td>7548-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Anisole anhydrous, 99.7%</td>
			<td>Sigma-Aldrich</td>
			<td>296295</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica nanoparticle</td>
			<td>Microparticles GmbH</td>
			<td>SiO2-R-0.7</td>
			<td>5% w/v aqueous suspension</td>
		</tr>
		<tr>
			<td>3-Aminopropyltrimethoxysilane, &#62;96.0%</td>
			<td>Tokyo Chemical Industry</td>
			<td>T1255</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide for HPLC, &#8805;99.7%</td>
			<td>Sigma-Aldrich</td>
			<td>34869</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino-terminated poly(ethylene glycol) methyl ether</td>
			<td>Polymer Source</td>
			<td>P16082-EGOCH3NH2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablet</td>
			<td>Takara</td>
			<td>T9181</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Calbiochem</td>
			<td>9480</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 8.0)</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Invitrogen</td>
			<td>AM9640G</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>VWR</td>
			<td>E109-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Biosesang</td>
			<td>D1037</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Merck</td>
			<td>535140-1MLCN</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromo phenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B5525-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 6.8)</td>
			<td>Biosolution</td>
			<td>BT033</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Biosolution</td>
			<td>BS003</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PKR Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>12297S</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32233</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Cell Signaling Technology</td>
			<td>2729S</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>Korea Cell Line Bank</td>
			<td>10002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>DAIHAN Scientific</td>
			<td>WUC-D10H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>BMBio</td>
			<td>BR2006A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge I</td>
			<td>Eppendorf</td>
			<td>5424 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge II</td>
			<td>LABOGENE</td>
			<td>1736R</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>FINEPCR</td>
			<td>ROTATOR/AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>DAIHAN Scientific</td>
			<td>ThermoStable OV-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel permeation chromatography (THF)</td>
			<td>Agilent Technologies</td>
			<td>1260 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray photoelectron spectrometer</td>
			<td>Thermo VG Scientific</td>
			<td>Sigma Probe</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic light scattering</td>
			<td>Malvern Instruments</td>
			<td>ZEN 3690</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of poly(pentafluorophenyl acrylate) functionalized sio2 beads for protein purification

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.

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...

Watch this Scientific Journal Video about Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification at JoVE.com

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

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.

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

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

This method can be used to prepare biomolecule-activated surfaces, which have applications in areas such as drug delivery, biological target detection, and bio separation. The main advantage of this technique is that the poly PFPA brushes are highly reactive with amines, and the immobilization of antibodies is achieved by incubation in buffer solution for a few hours. The implications of this technique extend toward the protein purification through immunopurification, as the immobilized antibody can successfully bind with target antigens.Though this method is demonstrate for antibody mobilization and protein purification, it can also be applied to other systems requiring biomolecular mobilization. To treat silicon dioxide beads with APTES, first obtain silicon dioxide particles in the form of a 5%weight volume aqueous suspension. Combine 0.8 milliliters of silicon dioxide suspension with 40 milligrams of APTES, and eight milliliters of methanol in a 20 milliliter scintillation vial equipped with a stir bar.Allow the reaction to proceed at room temperature for five hours with vigorous stirring. After five hours, transfer the solution to a conical tube. To isolate the APTES functionalized silicone dioxide beads, centrifuge the solution at 10, 000 G for five minutes.Then remove the supernatant. Now wash the beads by redispursing them in three milliliters of fresh methanol. Shake the tube by hand for mixing, but if necessary, improve the dispersion by sonication in a water bath for a few seconds.Centrifuge as before, and repeat the wash step one more time. Combine the methanol wash silicone dioxide beads with three milliliters of dimethyl sulfoxide, or DMSO. Shake the mixture by hand, or if necessary, sonicate for a few seconds until the beads are fully dispersed in the DMSO.Following centrifugation at before, remove the supernatant. Repeat the centrifugation step a final time to ensure complete solvent exchange from methanol to DMSO. To prepare the poly PFPA solution, dissolve 20 milligrams of poly PFPA in two milliliters of DMSO in a 20 milliliter scintillation vial.To prepare PEG solution, dissolve amine functionalized PEG in one milliliter of DMSO. Now transfer the PEG solution to the poly PFPA solution. React at room temperature for one hour with vigorous stirring.Transfer one milliliter of the APTES functionalized silicon dioxide beads suspended in DMSO into the PEG substituted poly PFPA solution. Allow the grafting between poly PFPA and APTES functionalized silicon dioxide beads to proceed at room temperature for one hour with vigorous stirring. Then isolate the beads by centrifugation at 10, 000 G for five minutes, followed by the removal of the supernatant.Wash the beads by adding three milliliters of DMSO, and mix by hand or with a few seconds of sonication. Centrifuge the beads as before. And remove the supernatant before repeating the DMSO wash twice.Wash the beads twice more with three milliliters of triple distilled water. Then mix by hand or with a few seconds of sonication. Centrifuge the beads as before and remove the supernatant.Finally dry the beads at 40 degrees celsius in a vacuum oven overnight. Add five milligrams of poly PFPA grafted silicon dioxide beads to a 1.5 milliliter microcentrifuge tube. Wash the beads by adding 800 microliters of PBS, and mix well by vortexing.Centrifuge the beads at 10, 000 G at room temperature for one minute. Remove the supernatant, and repeat the wash step three times. Now add 350 microliters of fresh PBS, 50 microliters of 0.1%PBST, and 6.67 micrograms of the antibody.Incubate approximately 20 hours on a rotator at four degrees celsius. The next day, wash the beads and centrifuge at 400 G and four degrees celsius for one minute. Then remove the supernatant and carefully add 400 microliters of lysis buffer.Gently resuspend the beads by pipetting up and down five times. Repeat this wash step three times. After the final wash, remove as much of the supernatant as possible.Prepare cells and lysis buffer as described in the text protocol. Then resuspend the cell pellet with 400 microliters of the lysis buffer. Sonicate the cells using an ultra sonicator.After sonication, vortex briefly. Then centrifuge the lysate at 20, 000 G at four degrees celsius for 10 minutes. Transfer the supernatant to a new, 1.5 milliliter centrifuge tube.To perform immunoprecipitation, transfer 300 microliters of cell lysate to previously prepared antibody immobilized poly PFPA grafted silicon dioxide beads. Retain 30 microliters of the cell lysate as the input sample in a new microcentrifuge tube. Incubate the lysate beads mixture for three hours on a rotator at four degrees celsius.Following incubation, centrifuge the mixture at 400 G at four degrees celsius for one minute. Remove the supernatant, and carefully add 400 microliters of wash buffer. Gently resuspend the beads by pipetting up and down about five times.After three washes, remove as much of the supernatant as possible. Add 30 microliters of 2x SDS loading dye to the beads and to the input sample. Then heat them for 10 minutes at 95 degrees celsius.After heating, analyze the sample using western blotting, or store the sample at minus 20 degrees celsius. The functionalized silica beads are examined by x-ray photoelectron spectroscopy, or XPS, to determine surface composition. Representative XPS data are shown here.Following APTES treatment, the nitrogen 1s peak associated with the amine group zone APTES is detected. Following poly PFPA treatment, the fluorine 1s peak associated with the PFP units on the polymer is detected. Protein kinase RNA activated, or PKR enrichment through immunoprecipitation is performed, and the alluded protein sample are analyzed using western blotting.As expected, beads immobilized with no antibody, or a non-specific antibody mixuture show no PKR recovery. The beads incubated with anti-PKR can successfully enrich PKR as indicated by the presence of a PKR band and the absence of GAPDH band. While attempting this procedure, it's important to remember that when comparing of systems, the IP experiments as well as the subsequent western blotting analysis should be done simultaneously.After its development, this technique can be used for the immobilization of different biomolecular or material substrate. Moreover, this method has the additional benefit of tuning Only surface property to be tailored to suit each application's need.

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