6 μmol of Active (SRS11-31) or inactive (SRS11-66) probes dissolved in 500 μL DMSO and 300mg of Profinity™ Epoxide Resin (Bio-Rad) were incubated in saturated sodium bicarbonate at 45 °C for 3 days. The conjugation reaction was ended by adding 120 μL of 1 M ethanolamine to the reaction mixture. The conjugated probe-beads were used for further protein pull-down assay.
Profinity epoxide resin
Manufactured by Bio-Rad
Sourced in United States
Profinity™ Epoxide Resin is a high-performance affinity chromatography resin designed for the purification of biomolecules. The resin features a proprietary epoxide-activated agarose support that enables the covalent immobilization of a wide range of ligands, including proteins, peptides, and other biomolecules. This allows for the efficient capture and purification of target molecules from complex samples.
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Lab products found in correlation
5 protocols using profinity epoxide resin
1
Affinity-based Protein Pulldown Assay
2
Immobilization of CFX on Epoxide Resin
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CFX was immobilized on Profinity™ Epoxide Resin (Bio-Rad). For this, 2 g dry resin was swollen in MQ-H2O, twice washed with MQ-H2O and vacuum-filtered. After a second wash with coupling buffer (50 mM KCl, 132 mM NaOH, pH 13.0), the resin was mixed 1:2 with 5 mM CFX solution in coupling buffer. The reaction was protected from light and incubated over night at room temperature (RT) on an H5600 rotator (Labnet). Afterwards, the resin was washed with MQ-H2O, vacuum-filtered and remaining active groups were blocked by incubation with 1 M ethanolamine (MEA) for 4 h. Finally, the CFX-coupled resin was washed according to the supplier's instructions with alternating buffer change from pH 4.0 (100 mM acetate, 500 mM NaCl) to pH 8.0 (100 mM phosphate, 500 mM NaCl). Lastly, the resin was washed with MQ-H2O and stored in 0.02% (w/v) NaN3 at 4°C in the dark for up to 3 months.
3
Protein Purification and Characterization Techniques
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Materials and reagents used in these experiments included bicinchoninic acid (Thermo Fisher, Rockford, IL, USA), diethylamine (Fluka, Buchs, Switzerland), ELISA 96-well microplates (Maxi Sorp, 442,404; Nunc, Roskilde, DK), Costar high-binding plates (9018; Corning, NY, USA), High-trap Protein G HR and Sepharose CL-4B (both from GE-Healthcare Life Sciences, Chicago, IL, USA), Profinity epoxide resin (Bio-Rad, Madrid, Spain), Tween-20 and ortho-phenylenediamine (OPD) (Sigma, St. Louis, MO, USA). Buffers were PBS (phosphate-buffered saline, pH 7.4), PBST (PBS with 0.05% Tween-20), PBSM blocking buffer (5% non-fat dry milk in PBS with 10 μg/L NaN3), and 0.1 M glycine-hydrochloride pH 2.5.
4
Affinity Purification of Protein Complexes
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Profinity epoxide resin (Bio-Rad) was swollen and washed in ddH2O and then resuspended in coupling buffer (0.1 N NaOH, pH 13.0). BCT (5 mg) was dissolved in coupling buffer and conjugated with Profinity epoxide resin at room temperature for 18 h. After washing with coupling buffer, remaining active epoxide groups were blocked with 1 M ethanolamine (pH 8.0) for 4 h. The BCT-conjugated beads were washed with coupling buffer, wash buffers (WB1, 100 mM acetate, 500 mM NaCl, pH 4.5; WB2, 100 mM Na2HPO4, 500 mM NaCl, pH 8.0), and PBS. The control unconjugated Profinity beads were prepared as described above without addition of BCT. The control or BCT-conjugated beads were incubated with cell lysates overnight and washed with FLAG lysis buffer. Bound proteins were detected with specific antibodies as indicated in figures.
5
Affinity-based Protein Pulldown Assay
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