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C18 ziptip micropipette tips

Manufactured by Merck Group
Sourced in United States

The C18 ZipTip micropipette tips are a type of solid-phase extraction (SPE) tip used for sample preparation in analytical applications. The tips contain a sorbent material based on C18 reversed-phase chemistry, which allows for the selective extraction and concentration of analytes from complex samples prior to analysis.

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5 protocols using c18 ziptip micropipette tips

1

Protein Reduction, Alkylation, and Trypsin Digestion

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A gel containing 100 μg of proteins was mixed with dithiothreitol (final concentration of 10 mM) and kept at 60 °C for 70 min [15 (link),16 (link)]. After the sample was cooled, iodine acetamide with a final concentration of 66 mM was added and reacted away from light at room temperature for 40 min [16 (link)].
After that, 200 μL NH4HCO3 solution (50 mM) was added to the filter and centrifuged at 14,000× g for 25 min. Finally, 1.25 μg of trypsin was added and the mixture incubated at 40 °C overnight [16 (link),17 (link)]. The digestion procedure was stopped by adding formic acid (10%), and digested peptides were collected by centrifuging at 14,000× g for 15 min. After desalting with the C18 tips, the collected peptides were vacuum-dried [17 (link),18 (link)]. After trypsin digestion, Ziptip C18 micropipette tips (Millipore) were used to purify the peptides prior to adding 0.1% formic acid for LC-MS/MS analysis.
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2

MALDI-TOF Mass Spectrometry Protocol

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Mass spectrometry analysis was performed with a 4800 Plus (Sciex) or Microflex (Bruker Daltonics) MALDI-TOF mass spectrometer using 0.25 M aqueous 3-hydroxypicolinic acid as the matrix and 10 mM diammonium citrate as an additive. Spectra were recorded for positive ions. Before analysis, the samples were desalted and concentrated using ZipTip C18 micropipette tips (Millipore).
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3

Quantitative Proteomics with TMT

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TmT duplex isobaric tags reagent set (TMT2) and trypsin for MS were purchased from Thermo Scientific (San Jose, CA, USA). EDTA-free protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN, USA). Phosphatase inhibitors were purchased from Pierce (Idaho, ID, USA). RIPA Lysis Buffer was purchased from Thermo Scientific (San Jose, CA, USA). The Bradford assay was purchased from Applygen (Beijing, China). C18 ZipTip micropipette tips were purchased from Millipore (Bedford, MA, USA). Generic chemicals were purchased from Sigma-Aldrich (St. Louis, USA). All of the chemicals and reagents used in this study were of analytical chromatography grade.
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4

Cell Lysis and Tryptic Digestion for Proteomics

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Cells harvested from culture flasks were centrifuged and rinsed twice with 50mM ammonium bicarbonate solution to remove residues of the culture medium. Subsequently, the sample was suspended in 1 mL of 50% methanol in a solution of 0.1% trifluoroacetic acid (TFA) in water, mixed for 1 min and incubated at −20 °C for 5 min. The procedure of mixing and incubation was repeated three times. Then, the sample was sonicated for 15 min and centrifuged. The supernatant was divided into three tubes and subjected to the protocol for in-solution tryptic digestion, according to modified Pierce Kit procedure (Thermo Scientific, USA). After overnight digestion, samples were purified and concentrated using C18 ZipTip micropipette tips (Millipore, Burlington, MA, USA) according to the protocol provided by the manufacturer.
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5

MALDI-TOF-MS Peptide Detection Using SPE

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The solid phase extraction technique in the form of pipette tips pre-packed with hydrophobic phase was use as a sample pretreatment method. Prior MALDI-TOF-MS analyses, samples were diluted 1:5 using 0.1% TFA in water. In order to detect low molecular weight peptides depletion method using C18 ZipTip micropipette tips (Millipore, Bedford, MA, USA) was performed according to the manufacturer’s instruction. The protocol includes 3 steps: binding of proteins and peptides, washing, and elution. Fifty percent ACN solution was used for elution of the samples fractions. The detailed procedure was described in our previous study [41 (link)].
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