Hierarchical coassembly of DNA–triptycene hybrid molecular building blocks and zinc protoporphyrin IX

• Rina Kumari,
• Sumit Singh,
• Mohan Monisha,
• Sourav Bhowmick,
• Anindya Roy,
• Neeladri Das and
• Prolay Das

Beilstein J. Nanotechnol. 2016, 7, 697–707, doi:10.3762/bjnano.7.62

• , Cecil, UK). Equimolar ratios of S1 DNA-TPA with S2 DNA-TPA were annealed in 50 µL of total volume of 10 mM sodium phosphate buffer, 10 mM magnesium chloride and 100 mM NaCl (pH 7.2) by heating to 90 °C and allowing the solution to cool slowly to 4 °C at a rate of 0.1 °C per minute over 4 h. Zn PpIX was

• been carried out in 100 µL annealing buffer (10 mM sodium phosphate, 75 mM NaCl and 5 mM MgCl2) at pH 7.5. The samples were transferred into microcuvettes (Hellma cell) and kept at 20 °C for 2 min prior to measurement. At least five measurements were performed for each sample at 20 °C at a scattering

Investigating organic multilayers by spectroscopic ellipsometry: specific and non-specific interactions of polyhistidine with NTA self-assembled monolayers

• Ilaria Solano,
• Pietro Parisse,
• Ornella Cavalleri,
• Federico Gramazio,
• Loredana Casalis and
• Maurizio Canepa

Beilstein J. Nanotechnol. 2016, 7, 544–553, doi:10.3762/bjnano.7.48

• . Ethylenediaminetetraacetic acid (EDTA), imidazole, tris(hydroxymethyl)aminomethane (Tris), NaCl and NiCl2 were purchased from Sigma-Aldrich. NTA SAM deposition and interaction with His6 Clean Arrandee samples or freshly stripped Ulman gold samples were incubated for 24 h in 15 μM NTA1 (NTA2) solution, using ethanol as

• solvent. For ex situ optical measurements the NTA/Au samples were rinsed with ethanol and dried with N2 flow. A subset of NTA/Au samples were transferred in a salt buffer solution (20 mM Tris and 150 mM NaCl, pH 7.4) for subsequent characterization and treatments. After a first optical characterization in

• nanografted areas have been then imaged in soft contact mode in ethanol or in saline buffer (Tris-HCl 20 mM, NaCl 150 mM, pH 7.4) with commercial cantilevers (CSC38 Mikromasch, k = 0.03 N/m) at the minimum force detectable (0.1 nN). Images and data were analyzed with XEI (Park Instruments) and Igor Pro

Hemolysin coregulated protein 1 as a molecular gluing unit for the assembly of nanoparticle hybrid structures

• Tuan Anh Pham,
• Andreas Schreiber,
• Elena V. Sturm (née Rosseeva),
• Stefan Schiller and
• Helmut Cölfen

Beilstein J. Nanotechnol. 2016, 7, 351–363, doi:10.3762/bjnano.7.32

• only takes place for the Hcp1_cys3-functionalized NPs, where the citrate-stabilized Au NPs remain stable as evidenced by their unchanged surface plasmon resonance at 520 nm. When the NaCl concentration exceeded 12 mM, a prompt color change to blue followed by precipitation of a blue solid was observed

• Figure 6, the Raman spectra of the pure Hcp1_cys3 Au NP sample with 2 equiv Hcp1_cys3 in 6 mM NaCl from Figure 2A and the Au Hcp1_cys3 network from Figure 3 and Figure 4 are shown. These samples represent 3 cases: a) pure/bare Hcp1_cys3 protein, b) Hcp1_cys3 adsorbed on the Au NP surface and c) Hcp1_cys3

• centrifugation of the solution at 40000 rpm. The supernatant was discarded and precipitate was collected and redispersed in MeOH. The procedure was repeated three times. The solid was dried in a vacuum oven and could be easily dispersed in water. The storage buffer (50 mM Tris, 500 mM NaCl, 150 mM imidazol, 10

Influence of calcium on ceramide-1-phosphate monolayers

• Joana S. L. Oliveira,
• Gerald Brezesinski,
• Alexandra Hill and
• Arne Gericke

Beilstein J. Nanotechnol. 2016, 7, 236–245, doi:10.3762/bjnano.7.22

• with and without calcium in the subphase. For pH 9, a solution of 10 mM borax buffer has been used, and for pH 4, a 10 mM citric buffer. Either 150 mM NaCl was added to mimic the physiological environment and 1 mM EDTA to chelate traces of divalent cations, or the EDTA was substituted by 1 mM CaCl2. On

• ) was purchased from Matreya, LLC (PA, USA). EDTA (≥99.4%), NaCl (≥99.5%), NaOH (≥99.5%), CaCl2 (≥99%) and HCl were purchased from Sigma Aldrich GmbH (Taufkirchen, Germany). Chloroform (≥99.9%) and citric acid monohydrate (≥99.5%) were purchased from Carl Roth GmbH (Karlsruhe, Germany) and methanol

• (≥99.9%) from VWR Chemicals (Fontenay-sous-Bois, France). Sodium tetraborate decahydrate (≥99.5%) was purchased from Merck KGaA (Darmstadt, Germany). NaCl was heated to 600 °C to remove potential organic impurities. All other chemicals and salts were used without further purification. For monolayer

Mismatch detection in DNA monolayers by atomic force microscopy and electrochemical impedance spectroscopy

• Maryse D. Nkoua Ngavouka,
• Pietro Capaldo,
• Elena Ambrosetti,
• Giacinto Scoles,
• Loredana Casalis and
• Pietro Parisse

Beilstein J. Nanotechnol. 2016, 7, 220–227, doi:10.3762/bjnano.7.20

• , the AFM tip is scanned at high load (approx. 100 nN) over the TOEG6 SAM, operating in a buffer solution (10 mM Tris-HCl, 1 mM EDTA, (hereafter TE), 1 M NaCl, pH 7.1) containing 5 μM thiolated ssDNA oligonucleotides. The applied load is sufficient to displace the TOEG6 molecules from the gold surface

• cantilevers (CSC38 Mikromasch, spring constant: 0.06 N/m) at 1 Hz scan rate, applying a force of 0.1 nN. Hybridization was monitored after the addition of the required target solutions (1 μM target in TE buffer 1 M NaCl) into the AFM liquid cell for 1 h. All DNA sequences used in the present work are listed

• on gold [23][29]. Initially the electrodes were wetted for 10 min with a drop of a high-ionic-strength buffer, TE 1 M NaCl, containing 1 μM thiolated ssDNA. In this way a low-density ssDNA SAM (about 2 × 1012 to 3 × 1012 molecules/cm2) was obtained [29]. After DNA-functionalization the devices were

3D solid supported inter-polyelectrolyte complexes obtained by the alternate deposition of poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate)

• Eduardo Guzmán,
• Armando Maestro,
• Sara Llamas,
• Jesús Álvarez-Rodríguez,
• Francisco Ortega,
• Ángel Maroto-Valiente and
• Ramón G. Rubio

Beilstein J. Nanotechnol. 2016, 7, 197–208, doi:10.3762/bjnano.7.18

• purification. The ionic strength of the solutions was controlled by adding NaCl (Sigma-Aldrich, purity > 99.9%). The water used for all the experiments was of Milli-Q quality (Millipore RG model). All the experiments were done at (298.1 ± 1.0) K. Layer-by-layer assembly In a similar manner as described in [8

• ]. Figure 8b shows that the role of the extrinsic compensation in PDADMAC layers is significantly enhanced with the increase in the ionic strength. This is associated to the strong screening effect of NaCl on the charge of PDADMAC. This implies that the amount of counterions associated with the PDADMAC

• [sodium]/[sulfur] occurs. This is a further confirmation of the reduced effect of NaCl on the adsorption of PSS for low and moderate ionic strengths. The different trends found for the ratios of [sodium]/[sulfur] and [chloride]/[nitrogen] agree with the conclusion obtained from ellipsometry. Conclusion

Single-molecule mechanics of protein-labelled DNA handles

• Vivek S. Jadhav,
• Dorothea Brüggemann,
• Florian Wruck and
• Martin Hegner

Beilstein J. Nanotechnol. 2016, 7, 138–148, doi:10.3762/bjnano.7.16

• examine various coupling ratios of PDHs to anti-DIG beads for the subsequent biotin-streptavidin binding in OT experiments. All experiments were also performed in TICO buffer with an increased Mg content of 12 mM (high Mg TICO). Control experiments were carried out in standard buffer (150 mM NaCl, 10 mM

Single pyrimidine discrimination during voltage-driven translocation of osmylated oligodeoxynucleotides via the α-hemolysin nanopore

• Yun Ding and
• Anastassia Kanavarioti

Beilstein J. Nanotechnol. 2016, 7, 91–101, doi:10.3762/bjnano.7.11

• were used for data acquisition and processing. The column was a 2 × 250 mm BioLC DNAPac® PA200 from Dionex used in conjunction with 25 mM Tris·HCl pH 7 buffer and a NaCl gradient; the column was maintained at 30 °C. This type of ion-exchange chromatography resolves oligos based on length and

Fabrication and characterization of novel multilayered structures by stereocomplexion of poly(D-lactic acid)/poly(L-lactic acid) and self-assembly of polyelectrolytes

• Elena Dellacasa,
• Li Zhao,
• Gesheng Yang,
• Laura Pastorino and
• Gleb B. Sukhorukov

Beilstein J. Nanotechnol. 2016, 7, 81–90, doi:10.3762/bjnano.7.10

• adsorption steps (15 min in duration) of anionic PSS (2 mg/mL in 0.5 M NaCl) and cationic PAH (2 mg/ml in Milli-Q water) followed. After each adsorption step, three washings in Milli-Q water (1500 rpm for 1 min) were carried out. Once the four bilayer structures were deposited, one layer of PLL (5 mg/mL in

Sonochemical co-deposition of antibacterial nanoparticles and dyes on textiles

• Ilana Perelshtein,
• Anat Lipovsky,
• Nina Perkas,
• Tzanko Tzanov and
• Aharon Gedanken

Beilstein J. Nanotechnol. 2016, 7, 1–8, doi:10.3762/bjnano.7.1

• , however, find only one report in which sonochemistry was used for the co-deposition of AgNPs and AgCl on TiO2 NPs to form Ag@TiO2 and Ag/AgCl@TiO2 [18]. The synthetic process revealed that in fact it was a one-step process combining Ag+, TiO2 NPs, and NaCl in ethylene glycol solution. The sonochemical

• the dye structure. The crystalline structure of the metal oxides was examined by XRD (Bruker D8). Leaching experiments Possible leaching of the MO and dyes from the coated textile was tested in saline solution (0.9 % NaCl). A piece of fabric (70 mg) was placed in 10 mL saline and kept at 40 °C for 72

• cultures of the bacteria were transferred into a nutrient broth (NB) medium (“Difco” Detroit, MI) and grown at 37 °C with aeration. When the cell number reached 106 CFU, the cells were harvested by centrifugation and washed twice with a 0.85 % NaCl solution at pH 6.5 (saline). The fabric (2 × 2 cm2) was

Effects of spin–orbit coupling and many-body correlations in STM transport through copper phthalocyanine

• Benjamin Siegert,
• Andrea Donarini and
• Milena Grifoni

Beilstein J. Nanotechnol. 2015, 6, 2452–2462, doi:10.3762/bjnano.6.254

• = 2.2 in order to fit our spectrum to the experiment of Swart et al. [14], which was carried out with CuPc on a NaCl(3 ML)/Cu(100) substrate with a workfunction of = 4.65 eV. With this, we find a triplet–singlet separation of the anionic ground state of 18 meV, which is in good agreement with

Green and energy-efficient methods for the production of metallic nanoparticles

• Mitra Naghdi,
• Mehrdad Taheran,
• Satinder K. Brar,
• M. Verma,
• R. Y. Surampalli and
• J. R. Valero

Beilstein J. Nanotechnol. 2015, 6, 2354–2376, doi:10.3762/bjnano.6.243

• stable in NaCl solution. However, they are aggregated in the presence of NaNO3 or NaH2PO4 [141]. Laudenslager et al. used CTS and carboxymethyl chitosan (CMC) as stabilizing agent for production of Pt, Au and Ag NPs. These two biopolymers gave similar size distributions, while CMC showed higher

• surface of NPs which is responsible for the electrostatic repulsion and consequently stability at wide range of pH (2–10) and electrolyte concentration (up to 10−2 M of NaCl) [63]. Thekkae Padil and Cernik used gum karaya (GK) to produce copper oxide (CuO) NPs from CuCl2 at 75 °C for 60 min. According to

Fabrication of hybrid graphene oxide/polyelectrolyte capsules by means of layer-by-layer assembly on erythrocyte cell templates

• Joseba Irigoyen,
• Nikolaos Politakos,
• Eleftheria Diamanti,
• Elena Rojas,
• Marco Marradi,
• Raquel Ledezma,
• Layza Arizmendi,
• J. Alberto Rodríguez,
• Ronald F. Ziolo and
• Sergio E. Moya

Beilstein J. Nanotechnol. 2015, 6, 2310–2318, doi:10.3762/bjnano.6.237

• homogenously charged surface prior to the GO deposition. The assembly of the polyelectrolytes in the first layers and between the GO was done in 0.5 M NaCl as in these salt conditions the polymers assemble in a denser and compact structure, due to the optimal coiled conformation of the PEs at this ionic

• the coated cell, thus reducing aggregation. The removal of the cell interior by NaOCl is based on the oxidation of the cell content, which is mainly proteins, which become defragmented and are easily eliminated during centrifugation and washing cycles with NaCl and water. Oxidation also affects the

• concentration of 2.5% in filtered PBS buffer. After fixation, they were centrifuged and washed in 0.9% NaCl several times at 10 °C and kept refrigerated. Graphene oxide Graphene oxide was prepared by strong acid oxidation of graphite using Hummer’s method [25] as modified in [26] where the dispersion was water

Synthesis, characterization and in vitro biocompatibility study of Au/TMC/Fe₃O₄ nanocomposites as a promising, nontoxic system for biomedical applications

• Hanieh Shirazi,
• Maryam Daneshpour,
• Soheila Kashanian and
• Kobra Omidfar

Beilstein J. Nanotechnol. 2015, 6, 1677–1689, doi:10.3762/bjnano.6.170

• purchased from Sigma (UK). T47D human ductal breast epithelial tumor cell line was obtained from American Type Culture Collection (ATCC) (USA). FeCl3·6H2O (99.0%), FeCl2·4H2O (99.0%), NaOH, and acetic acid were purchased from Acros Organics (USA). Analytical HCl, NaCl, D-glucose, glutaraldehyde, N

• and the solution was stirred for 24 h at 60 °C. The primary polymer was precipitated by means of an appropriate amount of acetone and then solved into NaCl (15%). The resulting suspension was dialyzed against deionized water for three days and finally freeze-dried, producing a pale yellow, TMC powder

• [28][51][52]. Polymer-coated Fe3O4 nanoparticles First, 0.2 g of the previously sonicated iron oxide nanoparticles were dispersed in a 25.5 mL solution containing NaCl (0.5 M) and SDS (0.025 M). Given the molar ratio of polymer and Fe3O4 (1:1), an appropriate amount of chitosan or TMC (in 2% acetic

Atomic force microscopy as analytical tool to study physico-mechanical properties of intestinal cells

• Christa Schimpel,
• Oliver Werzer,
• Eleonore Fröhlich,
• Gerd Leitinger,
• Markus Absenger-Novak,
• Birgit Teubl,
• Andreas Zimmer and
• Eva Roblegg

Beilstein J. Nanotechnol. 2015, 6, 1457–1466, doi:10.3762/bjnano.6.151

• as described earlier in literature [64]. In brief, cells were quickly rinsed in warm phosphate buffered saline (PBS; 0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4) and fixed with 4% formaldehyde in PBS for 15 min at room temperature (RT). Next, cells were washed with PBS and permeabilized for 5 min at

Scalable, high performance, enzymatic cathodes based on nanoimprint lithography

• Dmitry Pankratov,
• Richard Sundberg,
• Javier Sotres,
• Dmitry B. Suyatin,
• Ivan Maximov,
• Sergey Shleev and
• Lars Montelius

Beilstein J. Nanotechnol. 2015, 6, 1377–1384, doi:10.3762/bjnano.6.142

• gift from Amano Enzyme, Inc. (Nagoya, Japan). The specific activity of BOx, measured to be 140 U·mg−1, was determined using 5 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as an electron donor dissolved in phosphate buffered saline (PBS; 50 mM phosphate buffer containing 0.15 M NaCl

Probing fibronectin–antibody interactions using AFM force spectroscopy and lateral force microscopy

• Andrzej J. Kulik,
• Małgorzata Lekka,
• Kyumin Lee,
• Grazyna Pyka-Fościak and
• Wieslaw Nowak

Beilstein J. Nanotechnol. 2015, 6, 1164–1175, doi:10.3762/bjnano.6.118

• fibronectin isolated from human plasma. Other reagents Other reagents used in the experiments were: (a) phosphate buffered saline (PBS, ICN Biomedicals, pH 7.4, containing 10 mM of PO42−, 137 mM of NaCl and 27 mM of KCl) was used to prepare all protein solutions; (b) 3-aminopropyltriethoxysilane (APTES, Sigma

Stick–slip behaviour on Au(111) with adsorption of copper and sulfate

• Nikolay Podgaynyy,
• Sabine Wezisla,
• Christoph Molls,
• Shahid Iqbal and
• Helmut Baltruschat

Beilstein J. Nanotechnol. 2015, 6, 820–830, doi:10.3762/bjnano.6.85

• . When the tip is scanning across a monatomic step, friction is largely increased due to the Schwoebel barrier, as has been shown for the NaCl(001)/gas interfaces [22]. We observed the same effect at the Au(111)/electrolyte interfaces [10][13]. Correspondingly, we have shown that friction is higher when

• was observed by Meyer and coworkers [33]: Upon an increase in normal load on a NaCl(001) surface a transition to multiple slip was found. According to [34], who predicted such transitions from theory for low damping conditions and also observed it on HOPG, this process is based on energy minimisation

In situ observation of biotite (001) surface dissolution at pH 1 and 9.5 by advanced optical microscopy

• Chiara Cappelli,
• Daniel Lamarca-Irisarri,
• Jordi Camas,
• F. Javier Huertas and
• Alexander E. S. Van Driessche

Beilstein J. Nanotechnol. 2015, 6, 665–673, doi:10.3762/bjnano.6.67

• over a wide range of experimental conditions [3][5][6][19][45][46]. Hu et al. [45] observed the formation of “fibrous illite structures” when biotite reacted in 1 mol·L−1 NaCl solution and high temperature (acidic hydrothermal conditions). Shao et al. [47] observed the formation of fibrous illite

Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability

• Ognen Pop-Georgievski,
• Dana Kubies,
• Josef Zemek,
• Neda Neykova,
• Roman Demianchuk,
• Eliška Mázl Chánová,
• Miroslav Šlouf,
• Milan Houska and
• František Rypáček

Beilstein J. Nanotechnol. 2015, 6, 617–631, doi:10.3762/bjnano.6.63

• protocol based on the work of Rowley et al. [60]. Alginate was dissolved in mixture of 0.1 M MES and 0.15 M NaCl, at pH 5, at a concentration of 1 wt %. Next, EDC was added and the solution was stirred for 15 min. Afterwards, NHS was added and the solution was stirred for an additional 15 min until the

Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells

• Syeda Arooj,
• Samina Nazir,
• Akhtar Nadhman,
• Nafees Ahmad,
• Bakhtiar Muhammad,
• Ishaq Ahmad,
• Kehkashan Mazhar and
• Rashda Abbasi

Beilstein J. Nanotechnol. 2015, 6, 570–582, doi:10.3762/bjnano.6.59

• washed and harvested in ice-cold phosphate buffer saline at 4 °C. Cells were then lysed in cell lysis buffer (2.5 M NaCl, 10 mM Trizma-base at pH 10.0, 100 mM Na2EDTA, 1% sodium sarcosinate, 1% Triton X-100, 10% DMSO) and centrifuged at 15000g for 10 min at 4 °C. The supernatant was collected and

Green preparation and spectroscopic characterization of plasmonic silver nanoparticles using fruits as reducing agents

• Jes Ærøe Hyllested,
• Marta Espina Palanco,
• Nicolai Hagen,
• Klaus Bo Mogensen and
• Katrin Kneipp

Beilstein J. Nanotechnol. 2015, 6, 293–299, doi:10.3762/bjnano.6.27

• nanoparticles are different depending on the fruit used for preparation. The green preparation process can result in individual nanoparticles with a very poor tendency to form aggregates with narrow gaps even when aggregation is forced by the addition of NaCl. This explains only modest enhancement factors for

• silver nanoparticles [9], such as adenine, guanine, sodium chloride, failed. The extinction spectra in Figure 2a and the SEM image show that also NaCl at relatively high concentration does not force the formation of aggregates with subnanometer gaps as it had been obtained for other silver nanoparticles

• oranges and pineapples, scale bar is 100 nm. a) Extinction spectra of solutions of green silver nanoparticles made from orange extract before and after the addition of NaCl, final NaCl concentration was 0.2 M. The inset shows an aggregate formed by green nanoparticles. b) SERS spectrum of pMBA collected

Kelvin probe force microscopy in liquid using electrochemical force microscopy

• Liam Collins,
• Stephen Jesse,
• Jason I. Kilpatrick,
• Alexander Tselev,
• M. Baris Okatan,
• Sergei V. Kalinin and
• Brian J. Rodriguez

Beilstein J. Nanotechnol. 2015, 6, 201–214, doi:10.3762/bjnano.6.19

• measurements are demonstrated in ionically-active (polar isopropanol, milli-Q water and aqueous NaCl) and ionically-inactive (non-polar decane) liquids by electrochemical force microscopy (EcFM), a multidimensional (i.e., bias- and time-resolved) spectroscopy method. In the absence of mobile charges (ambient

• aqueous NaCl) environments. The measurement of bias- and time-dependent ion dynamics allows different electrokinetic phenomena to be separated and a set of environmental and measurement timescale requirements for determining CPD under conditions comparable with KPFM to be delineated. Finally, the

• , to explore the dependence of the ion concentration on the EcFM response, single-point EcFM measurements were performed as a function of NaCl concentration (Figure 4). For measurements performed in the presence of NaCl, the data shows a non-linear bias dependence of . In addition, bubble formation was

Functionalization of α-synuclein fibrils

• Simona Povilonienė,
• Vida Časaitė,
• Virginijus Bukauskas,
• Arūnas Šetkus,
• Juozas Staniulis and
• Rolandas Meškys

Beilstein J. Nanotechnol. 2015, 6, 124–133, doi:10.3762/bjnano.6.12

• ), containing 0.1 mM EDTA, 0.2 mM PMSF, and 500 mM NaCl. The cell lysate was heated at 100 °C for 10 min and subsequently centrifuged at 13200g for 20 min. The supernatant was dialyzed against 50 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, pH 8.0, and loaded into a HiTrap ANX column (GE Healthcare

• ) equilibrated in the same buffer. Proteins were eluted using a linear gradient of 0–1 M NaCl. Fractions containing α-SynC141 were pooled and additionally applied to a HiTrap Q XL column (GE Healthcare) and were identified by 15% SDS-PAGE. The α-Syn protein, expressed and purified in the same manner as α-SynC141

• , was used for control assays. Protein purity was evaluated by gel electrophoresis. Mass spectrometry verified the presence of the cysteine residue in the C terminal. α-SynC141 and α-Syn fibril formation Lyophilized proteins were dissolved in the buffer: 50 mM Tris-HCl, 100 mM NaCl, pH 7.5 to a final

The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice

• Denise Bargheer,
• Artur Giemsa,
• Barbara Freund,
• Markus Heine,
• Christian Waurisch,
• Gordon M. Stachowski,
• Stephen G. Hickey,
• Alexander Eychmüller,
• Jörg Heeren and
• Peter Nielsen

Beilstein J. Nanotechnol. 2015, 6, 111–123, doi:10.3762/bjnano.6.11

• normal diet of 260 µg Zn [35][36][37]. Size-exclusion chromatography (SEC) Similar to that previously described in [24], SEC was performed using a Superose-6 10/300 GL column (Amersham Bioscience, Munich, Germany) with buffer (10 mM tris(hydroxymethyl)aminomethane, 0.15 mM NaCl, 10 mM EDTA) at a flow