The lesion-containing as well as the lesion-free control and competitor genomes were prepared, following the previously published procedures20 (link). Briefly, the parental vector was digested with Nt.BstNBI to generate a gapped plasmid, followed by removal of the resultant 25-mer single-stranded ODN through annealing with a 25-mer complementary ODN that is in 100× excess. The gapped plasmid was then isolated from the mixture by using 100 kDa cutoff ultracentrifugal filter units (Millipore). The purified gapped plasmid was annealed with a 5′-phosphorylated 13-mer lesion-free ODN (5′-AATTGAGTCGATG-3′) and a 5′-phosphorylated 12-mer lesion-carrying or lesion-free control ODN, 5′-ATGGCGXGCTAT-3′ (X = α-dN, x-dN, or dN), followed by incubation with T4 DNA ligase and ATP in the ligation buffer (Figure 2 ).
Ultracentrifugal filter units
Manufactured by Merck Group
Sourced in United States
Ultracentrifugal filter units are a type of laboratory equipment used for the separation and concentration of macromolecules, such as proteins and nucleic acids, from complex samples. The core function of these units is to facilitate the rapid and efficient separation of molecules based on their size and density through the application of centrifugal force.
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Lab products found in correlation
Dc protein assay, by Bio-Rad (1 mentions)
Foetal bovine serum (fbs), by Gemini Bio (1 mentions)
Paclitaxel ptx, by Meilun (1 mentions)
Icg sulfo osu, by AAT Bioquest (1 mentions)
Potassium sodium tartrate tetrahydrate, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Protein a resin, by GenScript (1 mentions)
Cobas c 501 autoanalyzer, by Roche (1 mentions)
6 protocols using ultracentrifugal filter units
1
Constructing Lesion-Containing Genomes
2
Western Blot Analysis of VEC Expression
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This assay was carried out as described previously [43 (link)]. Briefly, HUVECs were seeded at 105 cells/well in 6 cm diameter dishes and grown for 3 days in EGM-2. Cells were then switched to EBM-2 for 24 h and exposed to either diluent or human PROS1 (10 µg/mL) for 24 h. Cell lysates were analyzed by SDS-PAGE on 10% polyacrylamide gels and Western blotting (20–30 μg of total protein lysate/lane) using anti-human VEC mouse antibody (BD Biosciences; Franklin Lakes, NJ, USA) directed against the C-terminal intracellular domain of VEC. For the measurement of protein content, we used the DC protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Conditioned media from untreated or human PROS1 stimulated cells (normalized to cell numbers) were collected, centrifuged to discard floating cells, and concentrated on ultra-centrifugal filter units (10 kDa cut-off) (Millipore Burlington, MA, USA) (Table 1 ). A volume of 20 to 30 μL of concentrated medium of each condition was analyzed by SDS-PAGE and Western blotting using the anti-VEC clone BV6 monoclonal mouse antibody (Table 1 ).
3
Fluorescent Dye Labeling Protocol
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The N-hydroxysuccinimidyl (NHS) esters of different fluorescent dyes were purchased as follows: BDP, Cy3.5, Cy5 and Cy7.5 from Lumiprobe Corporation; ICG-Sulfo-OSu from AAT Bioquest Inc; BDP650 NHS ester from Life Technologies. Ultracentrifugal filter units (MWCO = 100 kDa) were obtained from Merck Millipore. Foetal bovine serum (FBS) was purchased from Gemini Bio-products Inc. Cell culture media, penicillin-streptomycin and 0.25% trypsin-EDTA were obtained from M&C gene technology Ltd. Paclitaxel (PTX) was purchased from Dalian Meilun Biotechnology Co. Ltd. Monomers, such as 2-(diisopropylamino) ethyl methacrylate (DPA-MA) and 2-aminoethyl methacrylate (AMA) were purchased from Polysciences Company. Initiator V65 was purchased from Yuanye Biotechnology Co. Ltd. Other reagents and solvents were from Thermo Fisher Scientific or Sigma-Aldrich Inc.
4
Radiolabeling of Nanobodies with Technetium-99m
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The nanobody (code, 15.2m; molecular weight, 15 kDa) was obtained from a phage display library as described previously (20 (link),21 (link)). Fresh technetium-99 m (99mTc)-pertechnetate eluant was purchased from the China Institute of Atomic Energy (Beijing, China). Sodium borohydride, HCl, sodium potassium tartrate tetrahydrate, sodium carbonate, and fluorescent dyes (FITC and DAPI) were purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Silica gel plates were obtained from Yantai Jiangyou Silica Gel Development Co., Ltd. (Yantai, China). Carbon monoxide and carbon dioxide were purchased from Tianjin KUNTENG Gas Marketing Co. Ltd. (Tianjin, China). Ultracentrifugal filter units with a molecular weight cut-off of 3,000 Da were purchased from EMD Millipore (Billerica, MA, USA) and used as per the manufacturer's manual. RPMI-1640 medium, penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Human serum was obtained from the Tianjin Blood Center (Tianjin, China). All procedures using human sera were approved by and conducted in accordance with the regulations of the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Tianjin, China).
5
Antibody Purification with Protein A
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For antibody purification, filtered supernatants were loaded onto a column packed with protein A resin (GenScript). After washing the column with PBS, Abs were eluted with 20 mM glycine pH 2.8 and neutralized with 1 M Tris HCl pH 9.5. Abs were concentrated and buffer exchanged to PBS using Ultra Centrifugal filter units (Merck). Concentrations of purified Abs were determined by NanoDrop.
6
Labeling LDL with DiI Fluorescent Dye
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LDL was labeled with DiI perchlorate-conjugated LDL (Invitrogen, Waltham, MA, USA), applying a modified procedure of the method described in Teupser et al. [57 (link)]. A stock solution of DiI was prepared by dissolving 3 mg DiI in 1 mL DMSO and then was added to the LDL solution to yield a final ratio of 150 μg DiI to 1 mg LDL. Then, it was incubated for 18 h at 37 °C under dark conditions followed by five rounds of centrifugation at 3000–3500× g for 45 min, using ultra centrifugal filter units (15 mL; Merck, Darmstadt, Germany) in order to isolate the DiI-labeled LDL. Then, DiI-LDL was dialyzed against saline containing phosphate-buffered saline (PBS) and filter-sterilized (0.25 μm, Water Millex HV units). The ApoB concentrations of LDL and DiI-LDL were determined using commercial kits adapted to a COBAS c501 autoanalyzer (Roche Diagnostics, Minato City, Tokyo). The standard solutions of DiI were prepared in isopropanol with a concentration range of 0–110 ng/mL. The standard curve of DiI-LDL was prepared in saline with a concentration range of 0–1600 ng protein/mL. A spectrofluorometer with excitation and emission wavelengths set at 549 and 564 nm was used to obtain fluorescence measurements. The specific activity of DiI-LDL was finally obtained as the amount of DiI (ng) integrated into 1 μg of LDL.
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