Eluates were sent on dry ice to the Scripps Research Institute, La Jolla, California. Samples were thawed and 125 μL were removed. Urea (60.06 mg) was added, and after urea was dissolved, 6.25 μL of 100 mM TCEP was added. Samples were shaken at RT for 30 min. Iodoacetamide (IAA, 6 μL of 250 mM) was added and the samples were covered in aluminum foil and shaken at RT for 30 min. EndoLysC (Wako, 1 μg) was added and the reactions were shaken at 37 °C for 4 hours. Samples were diluted 4x with 100 mM Tris and 1 μg trypsin was added. Samples were shaken overnight at 37 °C and stored in the freezer.
Endolysc
Manufactured by Fujifilm
EndoLysC is a laboratory equipment product manufactured by Fujifilm. It is designed for the processing and analysis of endoluminal samples. The product's core function is to provide a reliable and efficient platform for the preparation and examination of endoluminal specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using endolysc
1
Protein Reduction, Alkylation, and Digestion
2
Protein Extraction and Digestion from HEK293 Cells
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HEK293 cells (5 × 5 ×
106) were harvested, washed 3 times in phosphate-buffered
saline (PBS), and resuspended in lysis buffer (8 M urea; 20 mM HEPES,
pH 8.0). Samples were sonicated by three pulses of 15 s, interspaced
by 1 min pauses on ice, at an intensity output of 15 W, and centrifuged
for 15 min at 20 000g at room temperature
to remove insoluble components. The protein concentration in the supernatants
of each sample was measured using a Bradford assay (Bio-Rad), and
equal protein amounts (500 μg each) were used for further analysis.
Proteins were reduced with 5 mM dithiothreitol (DTT) (Sigma-Aldrich)
for 30 min at 55 °C. Alkylation was performed by the addition
of 10 mM iodoacetamide (Sigma-Aldrich) for 15 min at room temperature
in the dark. The samples were diluted with 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES), pH 8.0, to a urea concentration of 4 M, and proteins
were pre-digested with 5 μg of endoLysC (Wako, 129–02541)
(1/100, w/w) for 4 h at 37 °C. All samples were further diluted
with 20 mM HEPES, pH 8.0, to a final urea concentration of 2 M, and
proteins were digested with 5 μg of trypsin (V5111, Promega)
(1/100, w/w) overnight at 37 °C. Peptides were then purified
on a SampliQ SPE C18 cartridge (Agilent), vacuum-dried, and kept at
−20 °C until further use.
106) were harvested, washed 3 times in phosphate-buffered
saline (PBS), and resuspended in lysis buffer (8 M urea; 20 mM HEPES,
pH 8.0). Samples were sonicated by three pulses of 15 s, interspaced
by 1 min pauses on ice, at an intensity output of 15 W, and centrifuged
for 15 min at 20 000g at room temperature
to remove insoluble components. The protein concentration in the supernatants
of each sample was measured using a Bradford assay (Bio-Rad), and
equal protein amounts (500 μg each) were used for further analysis.
Proteins were reduced with 5 mM dithiothreitol (DTT) (Sigma-Aldrich)
for 30 min at 55 °C. Alkylation was performed by the addition
of 10 mM iodoacetamide (Sigma-Aldrich) for 15 min at room temperature
in the dark. The samples were diluted with 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES), pH 8.0, to a urea concentration of 4 M, and proteins
were pre-digested with 5 μg of endoLysC (Wako, 129–02541)
(1/100, w/w) for 4 h at 37 °C. All samples were further diluted
with 20 mM HEPES, pH 8.0, to a final urea concentration of 2 M, and
proteins were digested with 5 μg of trypsin (V5111, Promega)
(1/100, w/w) overnight at 37 °C. Peptides were then purified
on a SampliQ SPE C18 cartridge (Agilent), vacuum-dried, and kept at
−20 °C until further use.
3
SILAC-Based Protein Quantification and Digestion
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The protein concentration of the SILAC cell lysates was determined using the bicin chonicic acid assay (Pierce). Digestion of the proteins was performed using the Filter-Aided Sample Preparation (FASP) method [108 (link)], for which equal amounts (900 μg) of mock- and virus-infected cell lysates were mixed and DTT was added to a final concentration of 50 mM, followed by a 5-min incubation at 70°C. Samples were loaded on two 15-ml 30 kDa Microcon filter devices (Millipore), which were washed twice with 8 M urea 0.1 M Tris pH 8.5, while cysteines were alkylated with 50 mM iodoacetamide in the same buffer. Samples were washed 3 times with 8 M urea, 0.1 M Tris pH 8. Proteins were digested overnight at room temperature using 20 ug endoLysC (Wako Pure Chemical Industries) in the same buffer per filter device. The sample was diluted fourfold with 50 mM ammonium bicarbonate pH 8.4 containing 20 ug trypsin (Worthington Chemical Corporation), and digested for 4 h at room temperature. Peptides were collected by centrifugation, acidified to a final percentage of 1% TFA, and desalted using solid phase extraction. Peptides were eluted in 20/80/0.1 (v/v/v) of milliQ/acetonitrile (ACN) (Actu-All Chemicals)/trifluoric acid (TFA) (Sigma-Aldrich).
4
Protein Extraction and Digestion for MS
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Protein extracts were centrifuged 5 min at 5 k×g to pellet any insoluble material. An aliquot of the supernatant containing 100 µg protein was transferred to a new tube, adjusted to 66 µl, and precipitated by following the first steps of the protein precipitation protocol from a Bio-Rad 2-D clean-up kit (Bio-Rad; #1,632,130). Precipitated protein was re-solubilized in 50 µl 8 M urea supplemented with 5 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM chloroacetamide (CAA) for reduction and alkylation, respectively. Samples were digested by a two-step procedure by addition of 1 µg endo-Lys C (Wako Pure Chemical Corporation) and incubated for 3 h at 22 °C before dilution with 50 mM ammonium bicarbonate to 1 M urea final concentration and continued digestion over night after addition of 2 µg proteomics-grade trypsin (Sigma, # T6567). The digestion was quenched by addition of 40 µl 10% trifluoroacetic acid (TFA) and the digests were stored at − 80 °C until further analysis.
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