For each conditioned media sample, the volume corresponding to 20 µg of protein was taken and supplemented with 20% sodium dodecyl sulfate (SDS) to a final concentration of 4%. The samples were boiled at 95 °C for 10 min while shaking at 800 rpm on a Thermoshaker (Eppendorf). The samples were reduced with 5 mM dithiothreitol for 30 min at room temperature followed by alkylation with 15 mM iodoacetamide at 50 °C for 30 min in the dark. Samples were processed using the single-pot solid-phase enhanced sample preparation (SP3) [10 (link),11 (link)]. In short, protein purification, digestion and peptide clean-up were performed using a KingFisher Flex System (Thermo Fisher Scientific) and carboxylate-modified magnetic particles (GE Life Sciences; GE65152105050250, GE45152105050250) after the manufacturer’s instructions. Resulting digest solution and water elution were combined, dried and re-solubilized in 15 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid).
Ge45152105050250
Manufactured by GE Healthcare
Sourced in United States
The GE45152105050250 is a laboratory equipment product. It is designed to perform specific functions within a laboratory setting. The core function of this equipment is to provide precise measurements and data collection capabilities for researchers and scientists. Further details about the intended use or specific features of this product are not available in this unbiased, factual response.
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3 protocols using ge45152105050250
1
Automated Protein Purification and Preparation
2
Magnetic Particle-Based Protein Purification
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Speedbead magnetic carboxylate-modified particles (GE45152105050250 and GE65152105050250, GE Healthcare, Chicago, IL) were combined in a 1:1 (v/v) ratio, washed three times with water, and resuspended in water at a final concentration of 50 mg/mL. Prepared particles were added to the protein sample in a 10:1 (w/w) particle-to-protein ratio, and chilled acetonitrile (4 °C) was added in a 4:1 (v/v) acetonitrile-to-protein ratio to induce binding to the particles (Figure S2 ). The protein sample was incubated on a Thermomixer at 24 °C and 1,000 rpm for 5 minutes to enhance particle-protein binding, then placed on a 1 T custom-magnetic rack for 5 minutes at room temperature. Magnetic particles with bound proteins were pulled to the side of the microcentrifuge tube and contaminants in the supernatant were removed as waste. The protein-particle mixture was washed with 80 % ethanol in water (v/v), placed on the magnetic rack for 5 minutes, and the contaminant-containing supernatant was removed as waste.15 (link)
3
Protein Extraction and Purification Protocol
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Cells were lysed in buffer containing 4% sodium dodecyl sulfate (SDS) before boiling at 95 °C for 10 min. Twenty µg of proteins was taken and reduced with 5 mM dithio-threitol for 30 min at room temperature followed by alkylation with 15 mM iodoacetamide at 50 °C for 30 min in the dark. Samples were processed using the single-pot solid-phase enhanced sample preparation (SP3) [36 (link),37 (link)]. Protein purification, digest and peptide clean-up were performed using the KingFisher Flex System (Thermo Fisher Scientific, Waltham, MA, USA) and Carboxylate-Modified Magnetic Particles (GE Life Sciences, Piscataway, NJ, USA; GE65152105050250, GE45152105050250), as described previously [38 (link)]. After the digest, peptides were re-solubilised in 15 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid).
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