Saccharomyces cerevisiae strains BY4742 and BY4743 (EUROSCARF) were grown at 30 °C in yeast extract peptone dextrose (YPD) media (10 g/l BactoYeast extract, 20 g/l BactoTM peptone (BD), 2% w/v glucose). Cells were grown to log phase (OD600 of 0.6), harvested by centrifugation at 1600 × g for 10 min at 4 °C, washed with cold Milli-Q water and then collected again by centrifugation at 10,000 × g for 5 min at 4 °C. Cells were lysed in 1% sodium deoxycholate, 10 mm TCEP, 40 mm CAA in 100 mm Tris pH 8.5, boiled for 10 min at 95 °C and sonicated for 3 min at 30% duty cycle and output control 3 (Branson Ultrasonics sonifier; model 250). Protein concentrations were determined by tryptophan fluorescence emission assay. Cell lysates were diluted 1:2 with Milli-Q water and digested by adding LysC (Wako Chemicals GmbH, ratio 1 μg LysC:50 μg sample protein) for 4 h at 37 °C, followed by adding again LysC (ratio 1:50) overnight at 37 °C. An equal volume of ethyl acetate acidified with 1% TFA was added to the solution, samples were vortexed for 2 min and digested peptides were purified with SDB-RPS StageTips as described in Kulak et al. (19 (link)). Peptide concentrations were determined using a NanoDrop spectrophotometer.
Lys c
Manufactured by Fujifilm
Sourced in Japan, United States, Germany
Lys-C is a protease enzyme used in proteomics research. It selectively cleaves proteins at the carboxyl side of lysine residues, which can be useful for generating peptide fragments for analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Promega (184 mentions)
Trypsin, by Merck Group (34 mentions)
Trypsin, by Thermo Fisher Scientific (27 mentions)
Sep pak c18 cartridge, by Waters Corporation (23 mentions)
Bca assay, by Thermo Fisher Scientific (18 mentions)
Sep pak, by Waters Corporation (16 mentions)
Sequencing grade trypsin, by Promega (12 mentions)
Iodoacetamide, by Merck Group (12 mentions)
Phosstop, by Roche (11 mentions)
Thermomixer, by Eppendorf (11 mentions)
Dithiothreitol (dtt), by Merck Group (11 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (9 mentions)
Microspin c18 column, by Nest Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Sep pak c18 column, by Waters Corporation (8 mentions)
Tandem mass tag isobaric reagents, by Thermo Fisher Scientific (6 mentions)
Sequencing grade modified trypsin, by Promega (6 mentions)
C18 cartridges, by Harvard Apparatus (6 mentions)
Orbitrap elite, by Thermo Fisher Scientific (5 mentions)
Bradford assay, by Bio-Rad (5 mentions)
369 protocols using lys c
1
Yeast Culture and Protein Extraction
2
Protein Lysis and Digestion for Mass Spec
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer containing 6 M GndHCl, 100 mM Tris (pH 8.0), 1 mM EDTA, 5 mM tris(2-carboxyethyl)phosphine (TCEP; C4706, Sigma Aldrich), 10 mM chloroacetamide (CAA; 22790, Sigma Aldrich). Lysis was followed by immediate sonication at 70% amplitude (Sonics) three times for 20 s. Samples were kept on ice between the sonication cycles. Samples were cleared by centrifugation at 14,000 × g for 15 min at 4 °C and the supernatant was collected. Protein concentration was measured using the Bradford protein assay and 8–10 mg total protein was used per pull-down. LysC (Wako; protein:LysC ratio = 100:1) was added to each sample and LysC digestion was performed for 4 h at room temperature. Samples were cooled down on ice and diluted to a GndHCl concentration of 2 M with a dilution buffer (100 mM Tris (pH 8.0), 1 mM EDTA, 5 mM TCEp, 10 mM CAA).
3
Protein Digestion and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1/10 volume of 55 mM DTT was added and incubated for 30 min at room temperature. This was followed by the addition of 120 mM iodoacetamide at a volume equal to the DTT solution and incubated for 30 min at room temperature in the dark. The samples were then diluted using 20 mM HEPES (pH 8) to a final concentration of 6 M urea. LysC (Wako) was added with a LysC to protein ratio of 1:100 and incubated at 37 °C overnight. The samples were further diluted to a final concentration of ~1 M urea using 20 mM HEPES (pH 8.0). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w). After 4 hours of digestion at 37 °C, the peptide solution was cleaned up using a C18 cartridge (3 M™ Empore™, 3 mL).
4
Protein Reduction, Alkylation, and Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 41
About 1 mg protein in 9 M urea lysis buffer was reduced by the addition of 1/10 volume of 55 mM DTT to the cleared cell supernatant and incubated for 30 min at room temperature, followed by addition of 120 mM iodoacetamide at a volume equal to the DTT solution and incubated for 30 min at room temperature in the dark. The samples were then diluted 1.5-fold using 20 mM Hepes (pH8) to a final concentration of 6 M urea. LysC (Wako 129-02451) was added with a LysC to protein ratio of 1:100 and incubated at 37° C. overnight. The samples were further diluted to a final concentration of ˜1 M urea using 20 mM Hepes (pH8). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w). After 4 hours of digestion at 37° C., the peptide solution was cleaned up using a C18 cartridge (Waters Sep-Pak Vac RC Cartridge, 100 mg Sorbent per Cartridge). The amount of peptide obtained was measured using Nanodrop (ThermoScientific).
5
Protein Reduction, Alkylation, and Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 41
About 1 mg protein in 9 M urea lysis buffer was reduced by the addition of 1/10 volume of 55 mM DTT to the cleared cell supernatant and incubated for 30 min at room temperature, followed by addition of 120 mM iodoacetamide at a volume equal to the DTT solution and incubated for 30 min at room temperature in the dark. The samples were then diluted 1.5-fold using 20 mM Hepes (pH8) to a final concentration of 6 M urea. LysC (Wako 129-02451) was added with a LysC to protein ratio of 1:100 and incubated at 37° C. overnight. The samples were further diluted to a final concentration of ˜1 M urea using 20 mM Hepes (pH8). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w). After 4 hours of digestion at 37° C., the peptide solution was cleaned up using a C18 cartridge (Waters Sep-Pak Vac R C Cartridge, 100 mg Sorbent per Cartridge). The amount of peptide obtained was measured using Nanodrop (ThermoScientific).
6
Protein Extraction and Proteolytic Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein crude extracts without pretreatment were directly reduced with 10 mM dithiothreitol (DTT, Wako) at 37°C for 30 min and then alkylated with 50 mM iodoacetamide (Wako) at 37°C in the dark for 30 min. Alkylated proteins were digested with Lys-C (1:50, w/w, Wako) for 3 hours followed by overnight digestion with trypsin at 37°C (1:50, w/w, Promega). The surfactants were removed by adding organic solvent as described previously (49) . Tryptic peptides were dissolved in 5% acetonitrile (ACN, Wako) and 0.1% trifluoroacetic acid (TFA, Wako) for desalting with a styrene-divinylbenzene copolymer (SDB-XC) solid-phase extraction cartridge (Empore, 3M). In the case of pretreated samples, protein extracts were dissolved and denatured in 8 M urea (Invitrogen), which was followed by protein reduction and alkylation at room temperature. The alkylated protein was digested with Lys-C for 3 hours and then diluted four times with 50 mM ammonium bicarbonate (Wako) for overnight digestion with trypsin at room temperature. The resulting peptide mixture was acidified with TFA and desalted with an SDB-XC solid-phase extraction cartridge.
7
Protein Extraction and Digestion Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On day 1,548, 15 ml of biomass was collected, pelleted by centrifugation at 7,942g and 4 °C for 20 min and stored at −80 °C. Biomass pellets were resuspended in 100 µl milliQ water and heated at 95 °C for 5 min with agitation before sonication for 10 min in a sonication bath (Branson 5800 Ultrasonic Cleaner). Samples were centrifuged at 14,000g for 20 min at 4 °C, and supernatant was transferred to a new Eppendorf tube as ‘soluble’ extracts and RapiGest SF (Waters) was added to a final concentration of 0.1% (v/v). Pellets were washed with 100 µl milliQ water before addition of 50 µl 2% (v/v) RapiGest SF solution and heated at 95 °C for 5 min. Samples were reduced with bond-breaker TCEP solution (Thermo Fisher Scientific) for 20 min before alkylation with 50 mM chloroacetamide for 20 min at room temperature in the dark. Enzymatic digestion was performed by incubating protein extracts overnight at 37 °C with 0.4 µg LysC (Wako Chemicals Europe) and 0.4 µg trypsin (Promega). RapiGest SF was removed by incubating tryptic digests in 0.5% trifluoroacetic acid at 37 °C for 40 min and subsequent centrifugation at 14,000g for 20 min at 4 °C. Samples were analysed both before and after salt-mediated organic solvent precipitation81 (link).
8
Protein Extraction and Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From each fraction, 90 µl was mixing with 30 µl of 5x Laemmli loading buffer. The fractions were analyzed in 12% SDS-Page standard gel followed by Coomassie staining (20 µl for the fractions, 3 µl for the lysate and 10 µl for the pellet). KhpB was detected by the αKhpB antibody (2, SY0917 (MA11010) SABC-serum) (rabbit, 1:10,000) and a secondary anti-rabbit-HRP (goat) conjugate antibody ( 1:10,000, Sigma-Aldrich, #31460). The rest of the samples was prepared for mass spectrometric analysis as previously described (Hör et al. 2020b (link)). Briefly, samples were homogenized using ultrasound. Debris were later removed by centrifugation and 20 µl of the cleared protein sample were spiked-in with 10 µl of UPS2 spike-in (Sigma-Aldrich) diluted in 250 µl of 1.25x protein loading buffer. The samples were then reduced in 50 mM DTT, 10 min at 70°C and alkylated with 120 mM iodoacetamide for 20 min at room temperature in the dark. The proteins were precipitated using four times their volume of acetone overnight at −20°C, and later on washed with acetone and dissolved in 50 µl of 8 M urea, 100 mM ammonium bicarbonate. Protein digestion into peptides was performed using Lys-C (Wako) for 2 h at 30°C following by overnight digestion by trypsin. Peptides were eluted with 60% acetonitrile/0.3% formic acid and stored at −20°C until LC-MS7MS analysis.
9
Affinity Purification of VAP Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant protein expression in E. coli and purification using plasmids encoding the MSP domain of VAP-A (8–212; WT and KD/MD mutant) and VAP-B (1–210; WT and KD/MD mutant) were previously described (Di Mattia et al, 2020 (link)). For protein pull-down, the affinity resin was prepared by incubating 100 μg of recombinant protein with 20 μl of nickel beads (PureProteome Nickel Magnetic Beads; Merck) in pull-down buffer PDB (50 mM Tris–HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM imidazole, cOmplete protease inhibitor cocktail [Roche], and PhosSTOP [Roche]). The beads were then washed three times with the same buffer. 8 × 108 HeLa cells were washed with 5 ml of TBS and lysed with 1 ml of PDB. After a 10-min incubation on ice, the protein extract was separated from cell debris by centrifugation (10 min; 9,500g; 4°C). The protein extract was mixed with VAP-coupled nickel beads and incubated for 2 h at 4°C under constant agitation. The beads were then washed three times with PDB, and proteins were eluted with Laemmli buffer. Proteins were precipitated with trichloroacetic acid and digested with Lys-C (Wako) and trypsin (Promega). Peptides were then analysed using Ultimate 3000 nano-RSLC (Thermo Fisher Scientific) coupled in-line with Orbitrap ELITE (Thermo Fisher Scientific).
10
Bacterial Proteome Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial pellets were lysed in 8 M urea. The protein concentration of each sample was measured using a Bradford assay (Bio-Rad) and equal protein amounts, each containing 250 μg total protein, were used for further analysis. Proteins in each sample were reduced by adding 5 mM (final concentration) dithiothreitol and samples were incubated for 30 min at 55°C. Proteins were then alkylated by the addition of 10 mM (final concentration) chloroacetamide for 15 min at room temperature in the dark. Each sample was then diluted with 20 mM HEPES (pH 8.0) to a urea concentration of 4 M, and the proteins were digested with 2.5 μg LysC (Wako) (1/100, wt/wt) for 4 h under agitation at 37°C. The samples were then further diluted to a urea concentration of 2 M with 20 mM HEPES (pH 8.0) and digested with 2.5 μg trypsin (Promega) (1/200, wt/wt) overnight at 37°C. Samples were acidified to 1% trifluoroacetic acid (TFA) and desalted on reverse-phase C18 OMIX tips (Pierce), all according to the manufacturer’s specifications. Purified peptides were then stored at −80°C until liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
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!