The preparation of SISPROT-based cartridges strictly followed ten steps. In brief, the SISPROT-based cartridges were constructed by assembling the top and bottom tips. The bottom tips were packed sequentially with three plugs of the C18 membrane (3 M Empore) and mixed-mode ion exchange beads (SCX : SAX = 1 : 1, Applied Biosystems). The quantity of the C18 membrane and mixed-mode ion exchange beads was adjusted based on the protein amount.
Empore
Manufactured by 3M
Sourced in United States
Empore is a line of laboratory filtration products manufactured by 3M. The core function of Empore is to provide efficient and reliable filtration for a variety of applications in scientific and analytical settings. Empore products are designed to meet the needs of researchers, scientists, and technicians working in various fields, including pharmaceutical, environmental, and life sciences.
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Lab products found in correlation
10plex isobaric label reagent set, by Thermo Fisher Scientific (2 mentions)
Xbridge peptide beh column, by Waters Corporation (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lightfield, by Teledyne (1 mentions)
Matlab r2017b, by MathWorks (1 mentions)
Labview, by National Instruments (1 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Lys c, by Fujifilm (1 mentions)
Thermofisher q exactive, by Thermo Fisher Scientific (1 mentions)
Luna c18 resin, by Phenomenex (1 mentions)
C18 sd sorbent, by 3M (1 mentions)
Waters nanoacquity hplc system, by Waters Corporation (1 mentions)
8 protocols using empore
1
SISPROT-based Cartridge Preparation
2
Near-Infrared Fluorescence Spectroscopy of DNA-SWCNTs
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Fluorescence emission spectra were collected using a custom built near-infrared optical setup on an inverted Nikon Eclipse Ti-E microscope (Nikon AG Instruments), as described previously 12, 36 . Measurements were recorded with LightField (Princeton Instruments) in combina-tion with a custom-built LabView (National Instruments) software for automation purposes.
An exposure time of 10 seconds and laser excitation with band width of 10 nm and relative power of 100% was used for all measurements, unless stated otherwise. was used to collect the nanotube emission between 900 -1400 nm. Spectral fitting was performed using a custom Python program.
Photoluminescence excitation (PLE) maps were acquired between 525 nm and 800 nm with a 5 nm step. 50 µL aliquots of DNA-and LNA-SWCNT solutions (10 mg/L) were added to a 384-well plate, which was then sealed (Empore, 3M) to avoid evaporation during the measurement. The results were analysed using a custom Matlab code (Matlab R2017b, Mathworks).
An exposure time of 10 seconds and laser excitation with band width of 10 nm and relative power of 100% was used for all measurements, unless stated otherwise. was used to collect the nanotube emission between 900 -1400 nm. Spectral fitting was performed using a custom Python program.
Photoluminescence excitation (PLE) maps were acquired between 525 nm and 800 nm with a 5 nm step. 50 µL aliquots of DNA-and LNA-SWCNT solutions (10 mg/L) were added to a 384-well plate, which was then sealed (Empore, 3M) to avoid evaporation during the measurement. The results were analysed using a custom Matlab code (Matlab R2017b, Mathworks).
3
Gamma Irradiation-Induced Phosphorylation Proteomics
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We irradiated the cells, placed in a flask using a 60Co gamma-ray source with a dose rate of 1.04 Gy∙min−1, to stimulate phosphorylation within the cell culture. After irradiation, we transferred the cells back into an incubator and cultivated further for one hour.
After irradiation, we lysed H1299 cells using lysis buffer with a composition of 50 mM ammonium bicarbonate, 1% sodium deoxycholate, and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA) by heating them to 100 °C for 5 min followed by cooling on ice and addition of 1.5 mM MgCl2 and 2.5 U∙mL−1 Benzonase® Nuclease (Sigma-Aldrich, St. Louis, MO, USA) to degrade all forms of DNA and RNA. We precipitated the obtained proteins using the chloroform/methanol precipitation [41 (link)], desalted them using C18 solid-phase extraction cartridges (3M™ Empore™, St. Paul, MN, USA), reduced by dithiothreitol, alkylated by iodoacetamide (both Sigma-Aldrich, St. Louis, MO, USA), and digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI, USA) overnight at 37 °C.
After irradiation, we lysed H1299 cells using lysis buffer with a composition of 50 mM ammonium bicarbonate, 1% sodium deoxycholate, and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA) by heating them to 100 °C for 5 min followed by cooling on ice and addition of 1.5 mM MgCl2 and 2.5 U∙mL−1 Benzonase® Nuclease (Sigma-Aldrich, St. Louis, MO, USA) to degrade all forms of DNA and RNA. We precipitated the obtained proteins using the chloroform/methanol precipitation [41 (link)], desalted them using C18 solid-phase extraction cartridges (3M™ Empore™, St. Paul, MN, USA), reduced by dithiothreitol, alkylated by iodoacetamide (both Sigma-Aldrich, St. Louis, MO, USA), and digested with trypsin (Sequencing Grade Modified Trypsin, Promega Corporation, Madison, WI, USA) overnight at 37 °C.
4
Quantitative Proteomic Analysis via FASP and TMT
5
Quantitative Proteomic Analysis via FASP and TMT
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Samples were quantified using a micro BCA protein assay kit (Thermo Fisher Scientific) and 200μg of each sample was processed and digested using the Filter Aided Sample Preparation (FASP) method 58 (link). The samples were then desalted using a 7 mm, 3 ml C18 SPE cartridge column (Empore™, 3M) and labelled with TMT 59 (link) 10plex™ Isobaric Label Reagent Set (Thermo Fisher Scientific) as per manufacturers instructions. After labelling, the peptides from the 10 samples were pooled together in equal proportion. The pooled sample was fractionated into 20 discrete fractions using high pH reverse phase chromatography 60 (link) on a XBridge peptide BEH column (130 Å, 3.5 µm 2.1 X 150 mm, Waters) using an Ultimate 3000 HPLC system (Thermo Scientific/Dionex). Column temperature was set to 20 °C. The peptides were separated using a mix of buffers A (10 mM ammonium formate in water, pH 10) and B (10 mM ammonium formate in 90% CH3CN, pH 10). The peptides were eluted from the column using a flow rate of 200 μl/min and a linear gradient from 5% to 60% buffer B over 60 min. The peptides eluted from the column were separated into 40 fractions prior to concatenation into 20 fractions based on the UV signal of each fraction. All the fractions were dried in a speedvac concentrator and resuspended in 10 μl 5% formic acid, then diluted to 1% prior to MS analysis.
6
Protein Digestion and Purification
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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).
7
SDB-RPS and HLB Disk Conditioning
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Two types of SDB-RPS disks were used, one from 3M™’s Empore™ (E-RPS) product line and the alternative was from Affinisep’s AttractSPE™ (A-RPS) product line. Furthermore, HLB divinylbenzene disks with hydrophilic moieties were also selected from Affinisep’s AttractSPE™ line. Each type of sampling disks measured 47 mm in diameter.
All disks were conditioned in batches according to Scheithauer (2015 ). Each type of disk was put into separate beakers filled with 20 mL of methanol per disk. After gently shaking for 30 min, methanol was decanted and solvent remains were removed by adding 20 mL of double-distilled water per disk. Thereafter, the water was decanted and fresh water (20 mL per disk) was added followed by gentle shaking for 30 min. Finally, disks were mounted into stainless steel (SST) housings (2 mm thickness) with a single-sided opening of 40 mm in diameter (Fig.S2 ) (Vermeirssen et al. 2012 (link)). Samplers were wrapped in aluminum foil and stored in double-distilled water until use.
All disks were conditioned in batches according to Scheithauer (2015 ). Each type of disk was put into separate beakers filled with 20 mL of methanol per disk. After gently shaking for 30 min, methanol was decanted and solvent remains were removed by adding 20 mL of double-distilled water per disk. Thereafter, the water was decanted and fresh water (20 mL per disk) was added followed by gentle shaking for 30 min. Finally, disks were mounted into stainless steel (SST) housings (2 mm thickness) with a single-sided opening of 40 mm in diameter (Fig.
8
Proteomics Sample Preparation and Analysis
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Samples (100 μL) were desalted and concentrated using 30 kDa Spin-X UF centrifugal concentrators (Corning Inc., Lowell, MA, USA). Each flowthrough was then acidified in formic acid and cleaned of contaminants by solid phase extraction (SPE) on an Empore 96-well disc plate with C18-SD sorbent (3M, St. Paul, MN, USA). Eluates were lyophilised and resuspended in 0.1% TFA for analysis.
Samples were analysed by nano LC–MS/MS with a Waters NanoACQUITY HPLC system (Waters Corporation, Milford, MA, USA) interfaced to a ThermoFisher Q Exactive (ThermoFisher Scientific Inc., Canoga Park, CA, USA). Peptides were loaded on a trapping column and eluted over a 75 μm analytical column with a 1 h gradient at a flow rate of 350 nL·min−1. Both columns were packed with Luna C18 resin (Phenomenex, Torrance, CA, USA). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 FWHM and 17,500 FWHM resolution, respectively. From the MS scan, the 15 most intense ions were selected for MS/MS.
Samples were analysed by nano LC–MS/MS with a Waters NanoACQUITY HPLC system (Waters Corporation, Milford, MA, USA) interfaced to a ThermoFisher Q Exactive (ThermoFisher Scientific Inc., Canoga Park, CA, USA). Peptides were loaded on a trapping column and eluted over a 75 μm analytical column with a 1 h gradient at a flow rate of 350 nL·min−1. Both columns were packed with Luna C18 resin (Phenomenex, Torrance, CA, USA). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 FWHM and 17,500 FWHM resolution, respectively. From the MS scan, the 15 most intense ions were selected for MS/MS.
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