Following the immunoprecipitation procedure, beads were washed twice with 1 ml of ice-cold NH4HCO3 to exchange buffer and remove detergent as described17 (link). Washed beads were resuspended in 50 μl 50 mM NH4HCO3 containing 20 ng μl−1 SOLu trypsin (Sigma) and incubated at 25 °C with vigorous shaking overnight. Beads were then pelleted and supernatants were transferred to the new tubes.
Solu trypsin
Manufactured by Merck Group
Sourced in United States
SOLu-Trypsin is a laboratory reagent used for the enzymatic digestion of proteins. It is a purified, recombinant trypsin solution that is specifically designed to effectively solubilize and dissociate proteins from cell cultures or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Lc ms grade acetonitrile, by Merck Group (3 mentions)
Iodoacetamid, by Merck Group (2 mentions)
α2 3 6 8 9 neuraminidase, by New England Biolabs (2 mentions)
Glycobuffer 1, by New England Biolabs (2 mentions)
Lc ms grade formic acid, by Merck Group (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Pierce c18 spin tips, by Thermo Fisher Scientific (1 mentions)
Dithiotreitol, by Merck Group (1 mentions)
Bovine fetuin, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Glu c, by Promega (1 mentions)
Micro s traps, by Protifi (1 mentions)
Rapigest sf, by Waters Corporation (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Omix c18 10μl tips, by Agilent Technologies (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Acetic acid lc ms grade, by Merck Group (1 mentions)
Lc ms grade water, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
8 protocols using solu trypsin
1
Trypsin-based Peptide Extraction
2
Protein Identification in E. coli Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify a protein composition in E. coli lysates, proteins from 5 μl of clarified lysates were precipitated with acetone. Four volumes of acetone chilled to −20 °C were added to clarified lysates followed by 1 h of incubation at −20 °C. Precipitates were collected by 10 min of centrifugation at 20,000g at 4 °C. Pellets were rinsed with 80% acetone and let dry in air for 15 min. Dried pellets were dissolved in 5 μl of 50 mM NH4HCO3, 8 M urea, then mixed with 50 μl of 50 mM NH4HCO3 containing 20 ng μl−1 SOLu trypsin (Sigma) and incubated overnight at 25 °C.
After the overnight incubation, digestion reactions were mixed with an equal volume 2% HFBA, incubated at room temperature for 5 min and clarified by 5 min of centrifugation at 16,000g. Peptides from supernatants were then desalted using Pierce C18 spin tips (Thermo) according to the manufacturer’s instructions and dried under a vacuum. Dried peptides were reconstituted in 0.1% formic acid and concentration was measured at 205 nm on a Nanodrop One (Thermo).
After the overnight incubation, digestion reactions were mixed with an equal volume 2% HFBA, incubated at room temperature for 5 min and clarified by 5 min of centrifugation at 16,000g. Peptides from supernatants were then desalted using Pierce C18 spin tips (Thermo) according to the manufacturer’s instructions and dried under a vacuum. Dried peptides were reconstituted in 0.1% formic acid and concentration was measured at 205 nm on a Nanodrop One (Thermo).
3
Glycoprotein Analysis by Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetonitrile (LC-MS grade), formic acid (LC-MS grade), iodoacetamid (purity ≥ 99%), dithiotreitol (purity ≥ 99%), sex hormone-binding globulin from human serum, bovine fetuin, and SOLu-Trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hemopexin and haptoglobin standards from human plasma were purchased from Athens Research and Technology, Inc. (Athens, GA, USA). Water (LC-MS grade), ammonium hydrogen carbonate (LC-MS grade), and acetic acid (LC-MS grade) were supplied by Merck (Darmstadt, Germany). α2–3,6,8,9 neuraminidase, and GlycoBuffer 1 were purchased from New England BioLabs (Ipswich, MA, USA).
4
Trypsin and GluC Digestion of Solubilized TpoR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50 μg of purified TpoR was solubilized in 4% SDS, 100 mM Tris pH 8.5 by boiling for 10 min at 95 °C then prepared for digestion using Micro S-traps (Protifi, USA) according to the manufacturer’s instructions. Briefly, samples were reduced with 10 mM DTT for 10 min at 95 °C and then alkylated with 40 mM IAA in the dark for 1 h. Samples were then split into two aliquots, acidified to 1.2% phosphoric acid and diluted with seven volumes of S-trap wash buffer (90% methanol, 100 mM Tetraethylammonium bromide pH 7.1) before being loaded onto S-traps and washed three times with S-trap wash buffer. Each aliquot was digested with either 5 μg of Solu-Trypsin (Sigma) or 5 μg of GluC (Promega) overnight at 37 °C before being collected by centrifugation with washes of 100 mM Tetraethylammonium bromide, followed by 0.2% formic acid, followed by 0.2% formic acid/50% acetonitrile. Samples were dried down and further cleaned up using C18 Stage70 (link),71 (link) tips to ensure the removal of any particulate matter.
5
Protein Extraction and Digestion Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets were lysed in 50 μl of 0.1% RapiGest SF (Waters) with repeated pipetting, vortexing, and probe sonication at 20 kHz. EDTA-free protease inhibitor cocktail (Roche) combined with Benzonase Nuclease (Fisher Scientific) was added prior to cell lysis to reduce proteolysis and digest nucleic acids. To remove cell debris, lysates were centrifuged at 16,000g and 4 °C for 10 min. Total protein of lysates was measured with Pierce BCA protein assay kit (Fisher Scientific). Proteins were denaturated, and disulfide bonds were reduced by 10 mM dithiothreitol at 70 °C for 15 min and alkylated with 20 mM iodoacetamide at room temperature (RT) in the dark for 45 min. Digestion was completed overnight at 37 °C using recombinant dimethylated SOLu-trypsin (Sigma-Aldrich) with a trypsin:protein ratio 1:20. Trifluoroacetic acid (1%) was added to cleave and precipitate RapiGest SF, and 1 μl of 0.4 M L-methionine was added to limit methionine oxidation during storage. OMIX C18 10 μl tips (Agilent Technologies) were used for desalting and microextraction of tryptic peptides. Finally, samples were diluted in 5% acetonitrile with 0.1% formic acid.
6
Glycoprotein Analysis by LC-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetonitrile (LC-MS grade), formic acid (LC-MS grade), iodoacetamid (purity ≥ 99%), dithiotreitol (purity ≥ 99%), and SOLu-Trypsin were purchased from Sigma-Aldrich (St.Louis, MO, USA), as well as the IgG standard from human serum (UniProtKB ID, IgG1, P01857; IgG2, P01859). The hemopexin standard from human plasma (P02790) was purchased from Athens Research and Technology, Inc. (Athens, GA, USA). Water (LC-MS grade), ammonium hydrogen carbonate (LC-MS grade), and acetic acid (LC-MS grade) were supplied by Merck (Darmstadt, Germany). α2–3,6,8,9 neuraminidase (P0722S), and GlycoBuffer 1 was purchased from New England BioLabs (Ipswich, MA, USA).
7
Rapid Protein Digestion and LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein pellets were reconstituted in Rapid Digest Buffer (100 µL, Sigma-Aldrich, St. Louis, MO) and sonicated for 5 min before the addition of SOLu-trypsin (5 µg, Sigma-Aldrich). Digestion was performed for 1 h (cohorts 1 and 2) or 2 h (cohort 3) at 60 °C, quenched with formic acid and 80 µL transferred to a 96-well plate for LC–MS/MS.
8
Glycoprotein Purification and Enzymatic Digestion
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!