To increase the depth of proteome coverage, immunodepletion of highly abundant proteins was performed as previously described [7 ]. For CSF samples, 130 μl was incubated with equal volume (130 μl) of High Select Top14 Abundant Protein Depletion Resin (Thermo Fisher Scientific, A36372) at room temperature in centrifuge columns (Thermo Fisher Scientific, A89868). After 15 min of mixing with gentle rotation, the samples were centrifuged at 1000×g for 2 min. Sample flow-through was concentrated with a 3K Ultra Centrifugal Filter Device (Millipore, UFC500396) by centrifugation at 14,000×g for 30 min, and then the immunodepleted samples were diluted to equal volumes of 75 μl with phosphate-buffered saline. Immunodepleted CSF (60 μl) was then digested with LysC and trypsin. Briefly, the samples were reduced and alkylated with 1.2 μl of 0.5 M TCEP and 3 μl of 0.8 M CAA at 90°C for 10 min, followed by water bath sonication for 15 min. Samples were diluted with 193 μl of 8 M urea buffer [8 M urea in 10 mM Tris, 100 mM NaH2PO4 (pH 8.5)] to a final concentration of 6 M urea. LysC (4.5 μg) was used for overnight digestion at room temperature. Samples were then diluted to 1 M urea with 50 mM ABC. Trypsin (4.5 μg) was then added, and the samples were subsequently incubated for 12 h. The digestion was then stopped by adding final concentration of 1% FA and 0.1% TFA.
3k ultra centrifugal filter device
Manufactured by Merck Group
Sourced in United States
The 3K Ultra Centrifugal Filter Device is a lab equipment product offered by Merck Group. It is a centrifugal filtration device designed to separate and concentrate macromolecules, such as proteins, from solutions or suspensions. The device operates at high speed to efficiently remove unwanted components from the sample.
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2 protocols using 3k ultra centrifugal filter device
1
Immunodepletion-Based Proteome Profiling
2
In vitro Angiogenesis Assay of VEGF
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The angiogenic activity of VEGF produced by the tested cells was analyzed using the In vitro Angiogenesis Assay Kit (Chemicon, Temecula, CA, USA) as described previously.31 (link) Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 with 20% FCS, 50 U ml−1 penicillin, 50 μg ml−1 streptomycin sulfate, 25 μg ml−1 endothelial cell growth supplement, 100 μg ml−1 heparin, 2 mM sodium pyruvate and 1 mM HEPES at pH 7.4. HUVECs were serum starved in EGM-2 medium for 8 h at 37 °C. The supernatant of MCF-7 cells (conditioned medium of cultured cells) was collected and concentrated 10-fold using a 3 K Ultra centrifugal filter device (Millipore, Billerica, MA, USA). ECMatrix solution was mixed with ECMatrix diluent buffer, distributed to a 96-well plate and allowed to solidify at 37 °C for 1 h. The serum-starved HUVECs were resuspended with the concentrated supernatant (conditioned medium) and added to ECMatrix-coated 96-well plates, followed by incubation for 12 h. HUVEC capillary tube formation was inspected under an inverted light microscope and measured by counting the branch points in several random fields of view per well (averaged values). The results are taken from the experiment in triplicate of four independent experiments.
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