The lysates of the equivalent to 4 × 108 purified sEV were processed according to the sensitive Sp3 protocol [19 (link)]. The cysteine residues were reduced in 100 mM DTT and alkylated in 100 mM iodoacetamide (Acros Organics). 20 ug of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed two times with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured beads proteins were digested overnight at 37 °C under vigorous shaking (1200 rpm, Eppendorf Thermomixer) with 0.5 ug Trypsin/LysC (MS grade, Promega) prepared in 25 mM Ammonium bicarbonate. Next day, the supernatants were collected and the peptides were purified using a modified Sp3 clean-up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated, and the peptide concentration was determined through absorbance at 280 nm measurement using a nanodrop instrument.
Seramag carboxylate modified beads
Manufactured by GE Healthcare
Sourced in United States
SeraMag carboxylate-modified beads are a type of magnetic bead used for various laboratory applications. These beads have a carboxylate surface modification, which can be used to facilitate the separation, isolation, or purification of biomolecules such as proteins, nucleic acids, and cells. The magnetic properties of the beads allow for easy manipulation and separation using a magnetic field.
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Lab products found in correlation
6 protocols using seramag carboxylate modified beads
1
Sensitive Sp3 Protein Cleanup and Digestion
2
Sensitive Sp3 Protein Digestion and Cleanup
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The lysed samples were processed according to the sensitive Sp3 protocol [32 (link)]. The cysteine residues were reduced in 100 mM DTT and alkylated in 200 mM iodoacetamide (Acros Organics). 20 ug of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed two times with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured on-beads proteins were digested overnight at 37 °C under vigorous shaking (1200 rpm, Eppendorf Thermomixer) with 0.5 ug Trypsin/LysC (MS grade, Promega) prepared in 25 mM ammonium bicarbonate. Next day, the supernatants were collected, and the peptides were purified using a modified Sp3 clean up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated and the peptide concentration was determined through absorbance at 280-nm measurement using a nanodrop instrument.
3
Axon Proteomics Sample Preparation Protocol
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Axons were harvested by the addition of lysis buffer (1% SDC, 0.1% SDS, 100mM TrisHCl ph 8.5, 10mM DTT, 1x protease inhibitor EDTA free). Samples were supplemented with 25 units Benzonase nuclease (Merck), and lysed in a Bioruptor (Diagenode) for 5 minutes (cycle 30/30, 4°C). Alkylation was performed by addition of 30 mM Chloroacetamide followed by incubation in the dark for 30 min. Protein clean-up, digestion and peptide clean-up were performed using a modified version of the ultrasensitive sample preparation protocol SP3 (Hughes et al., 2014 (link)). In brief, 5 μL of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag Carboxylate-Modified beads, GE Life Sciences) were added to each sample. Acidified acetonitrile was added to achieve a final fraction of organic solvent of 50%. Beads were incubated for 10 min to allow complete binding of proteins to the beads. Protein clean-up was performed by subsequent wash with 70% Ethanol and once with Acetonitrile. For digestion, 0.1 μg sequencing grade Trypsin/LysC (Promega) was added and digestion was performed at 37°C for 16 h. Peptides were eluted with 9 μL 5% DMSO. 1 μL 10% formic acid was added and samples were stored at −20°C prior to MS analysis.
4
Ultrasensitive Axonal Protein Preparation
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Axons were harvested by addition of lysis buffer (1% SDC, 0.1% SDS, 100 mM TrisHCl ph 8.5, 10 mM DTT, 1x protease inhibitor EDTA free). Samples were supplemented with 25 units Benzonase nuclease (Merck), and lysed in a Bioruptor (Diagenode) for 5 min (cycle 30/30, 4°C). Alkylation was performed by addition of 30 mM Chloroacetamide followed by incubation in the dark for 30 min. Protein clean-up, digestion, and peptide clean-up were performed using a modified version of the recently developed ultrasensitive sample preparation protocol SP3 (Hughes et al., 2014 (link)). In brief, 5 μL of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag Carboxylate-Modified beads, GE Life Sciences) were added to each sample. Acidified acetonitrile was added to achieve a final fraction of organic solvent of 50%. Beads were incubated for 10 min to allow complete binding of proteins to the beads. Protein clean-up was performed by subsequent wash with 70% Ethanol and once with Acetonitrile. For digestion, 0.1 μg sequencing grade Trypsin/LysC (Promega) was added and digestion was performed at 37°C for 16 hr. Peptides were eluted with 9 μL 5% DMSO. 1 μL 10% formic acid was added and samples were stored at −20°C prior MS analysis.
5
Sensitive Sp3-based Protein Extraction
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The protein extracts, obtained from three representative collection sites in Naxos and three in Lakoma, with three biological replicates each one at harvest and at post-harvest, were processed according to the sensitive Sp3 protocol. The cysteine residues were reduced in 100 mM DTT and alkylated in 200 mM iodoacetamide (Acros Organics). 20 μg of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed twice with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured-on beads proteins were digested overnight at 37°C under vigorous shaking (1,200 rpm, Eppendorf Thermomixer) with 1 μg Trypsin/LysC (MS grade, Promega) prepared in 25 mM Ammonium bicarbonate. The next day, the supernatants were collected, and the peptides were purified using a modified Sp3 clean up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated and the peptide concentration was determined through absorbance at 280 nm measurement using a nanodrop instrument.
6
Guanidium Thiocyanate DNA Extraction
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A modified version of the guanidium thiocyanate protocol, described by Pitcher, Saunders & Owen (1989) (link), was used for DNA extraction (Pitcher, Saunders & Owen, 1989 (link)). After, samples were purified with SeraMag Carboxylate Modified beads® (GE Healthcare Life Sciences, Marlborough, MA, USA) in a binding buffer (10 mM Tris base, 1 mM EDTA, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 8.0), washed in 70% ethanol and eluted in TE buffer. Quality control was performed by Qubit® quantification and Nanodrop® absorbance determination.
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