Chemistry
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Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification
Chapters
Summary November 19th, 2018
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.
Transcript
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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