AFLP fingerprinting was performed with usually eight to ten individuals per population (Supplementary Table S1 ), using C. microcalyx (Boiss.) Wettst. and C. pubescens (C.Presl) Cufod. as outgroup taxa. AFLP profiles were generated following established protocols93 (link) with modifications94 ,95 (link). Three blanks (DNA replaced by water) were included to test for contamination, and 32 samples (10.5%) were used as replicates between PCR batches to test the reproducibility of AFLP fingerprinting. Based on an initial primer trial the following three selective primer combinations were chosen for selective PCR (fluorescent dye in brackets): EcoRI (6-FAM)ACA/MseI-CAT, EcoRI (VIC)AGG/MseI-CAC, and EcoRI (NED)AAC/MseI-CAG (6-FAM labelled primers: Sigma-Aldrich; NED and VIC labelled primers: Applied Biosystems). Selective PCR products were purified using Sephadex G-50 Fine (GE Healthcare Bio-Sciences, Uppsala, Sweden) applied to a MultiScreen-HV plate (Millipore, Molsheim, France) in three steps of 200 μl each and packed at 600 g for 1, 1 and 5 min, respectively. Then 0.8 µl of the elution product was mixed with 10 µl formamide (Applied Biosystems) and 0.125 µl GeneScan 500 ROX (Applied Biosystems) and run on an ABI 3130 automated capillary sequencer at the Department of Botany & Biodiversity Research of the University of Vienna, Austria.
Sephadex g 50 fine
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
Sephadex G-50 Fine is a gel filtration medium used for the separation and purification of molecules based on their size. It consists of small, porous beads made of cross-linked dextran, which allow smaller molecules to penetrate the pores while larger molecules are excluded, enabling their separation.
Automatically generated - may contain errors
Lab products found in correlation
Exosap it, by Thermo Fisher Scientific (2 mentions)
Sequencing primer, by Merck Group (2 mentions)
35s sulfate, by Hartmann Analytic (2 mentions)
Genescan 500 rox, by Thermo Fisher Scientific (1 mentions)
Formamide, by Thermo Fisher Scientific (1 mentions)
Abi bigdye terminator chemistry, by Thermo Fisher Scientific (1 mentions)
Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Dichloromethane dcm, by Thermo Fisher Scientific (1 mentions)
Recombinant protein a g, by Thermo Fisher Scientific (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Tetramethylrhodamine 5 maleimide, by Merck Group (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
16 protocols using sephadex g 50 fine
1
AFLP Fingerprinting Protocol for Plant Taxa
2
Sanger Sequencing of FCGR3A Gene
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Amplicons obtained from all 76 DNA samples were purified by ExoSAP-IT (Affymetrix, Santa Clara, California) following the manufacturer’s protocol.
Purified amplicons were sequenced using ABI BigDye Terminator Chemistry (Life Technologies) and an ABI 3730 sequencer (Life Technologies) with a forward and a reverse sequencing primer. For FCGR3A gene sequencing using Sanger, several sequencing primers were used to cover different locations in the gene. The sequencing mixture consisted of 1 µl purified PCR product, 0.5 µl sequencing primer (5 pmol, Sigma-Aldrich), 1 µl of BigDye Terminator v1.1 mix, 1.5 µl 5x Big Dye Terminator sequencing buffer and 6 µl distilled water. The PCR program consisted of: 1 minute at 95 °C, followed by 25 cycles of 10 seconds at 95 °C, 5 seconds at 50 °C, and 4 minutes at 60 °C. Successively, the mixtures were purified by Sephadex G-50 Fine (GE Healthcare Life Sciences, Little Chalfont, UK) and placed in the ABI 3730 sequencer for capillary electrophoresis sequencing. The chromatograms were aligned with a reference sequence obtained from the 1KGP and analysed using DNASTAR Lasergene SeqMan Pro (DNASTAR Lasergene, Madison, Wisconsin).
Purified amplicons were sequenced using ABI BigDye Terminator Chemistry (Life Technologies) and an ABI 3730 sequencer (Life Technologies) with a forward and a reverse sequencing primer. For FCGR3A gene sequencing using Sanger, several sequencing primers were used to cover different locations in the gene. The sequencing mixture consisted of 1 µl purified PCR product, 0.5 µl sequencing primer (5 pmol, Sigma-Aldrich), 1 µl of BigDye Terminator v1.1 mix, 1.5 µl 5x Big Dye Terminator sequencing buffer and 6 µl distilled water. The PCR program consisted of: 1 minute at 95 °C, followed by 25 cycles of 10 seconds at 95 °C, 5 seconds at 50 °C, and 4 minutes at 60 °C. Successively, the mixtures were purified by Sephadex G-50 Fine (GE Healthcare Life Sciences, Little Chalfont, UK) and placed in the ABI 3730 sequencer for capillary electrophoresis sequencing. The chromatograms were aligned with a reference sequence obtained from the 1KGP and analysed using DNASTAR Lasergene SeqMan Pro (DNASTAR Lasergene, Madison, Wisconsin).
3
Metabolic Labeling of Proteoglycans in HUVEC
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HUVEC cultures of the desired density were metabolically labeled with 0.1 mCi/ml 35S-sulfate (Hartmann Analytic) in RPMI-1640 sulfate free medium (GIBCO Invitrogen) added 5 mM L-glutamine (Sigma) and with FCS reduced from 7 to 2% to increase labeling efficiency. After labeling for 24 hours, the culture medium was collected. The cells were washed in PBS and harvested in either lysis buffer (4.0 M guanidine-HCl, 0.1 M acetate buffer pH 6.5, 2% Triton X-100) or RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% Na-deoxycholate, 10 mM EDTA, 10 mM Na4P2O7 and phosphatase inhibitor tablet freshly added). In order to remove unincorporated 35S-sulfate, samples were subjected to Sephadex G50 fine (GE Healthcare) gel chromatography in buffer (0.05 M Tris-HCl, 0.05 M NaCl, pH 8). The 35S -macromolecules were eluted in the void volume, while smaller molecules remained associated with the column. The amount of 35S-sulfate incorporated in newly synthesized 35S -macromolecules was determined by scintillation counting in triplicates. 35S-macromolecules in HUVEC are almost exclusively comprised of PGs [16 (link)]. Protein content of cell fractions was determined in RIPA-lysates prior to G50 fine gel chromatography or in guanidine-lysates after changing the buffer on the G50 fine column.
4
Radiolabeling and Purification of Sulfated Macromolecules
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Human myoblasts or myotubes were incubated in 100 μCi/mL (35S)sulfate (Hartmann Analytic GmbH, Braunschweig, Germany) in standard medium for 24 h. Conditioned media were collected and centrifuged to remove cell debris and the cell fractions were lysed in Hepes buffer (pH 7.4) with 1% Triton X-100. To remove unincorporated (35S)sulfate and to recover (35S)sulfated macromolecules, medium and cell lysates were subjected to Sephadex™ G-50 Fine (GE Healthcare, Little Chalfont, UK) gel chromatography (Meen et al. 2011 (link)). The sulfated macromolecules were eluted in the void volume with 0.05 mol/L Tris–HCl, 0.05 mol/L NaCl, pH 8. The amount of (35S)macromolecules isolated was quantified by scintillation counting. Samples were concentrated using Amicon Ultra centrifugal tubes with a molecular mass cut off at 3 kDa and the protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific).
5
Synthesis and Purification of Gold Nanoparticles
6
Labeling Monoclonal Antibodies with In-111
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The mAbs were conjugated with p-SCN-CHX-A’’-DTPA according to previous reports.18 (link) Briefly, mAbs (5 mg/mL) in 0.1 M borate buffer (pH 8.5) were incubated with a solution of p-SCN-CHX-A’’-DTPA (5 mg/mL) in 0.1 M borate buffer (pH 8.5) at 37°C for 16 hours. The number of p-SCN-CHX-A’’-DTPA introduced per molecule of IgG was 1.5–1.8, as determined by thin-layer chromatography (TLC). The conjugate was purified via the centrifuged column procedure using Sephadex G-50 Fine (GE Healthcare, 17-0042-01), equilibrated and eluted with 0.25 M acetate buffer (pH 5.5). [In-111]InCl3 was added to 1 M acetate buffer (pH 5.5) and incubated for 5 min at 25°C. Each conjugate was added to the solution and the mixture was incubated at 37°C for 1 hour, followed by addition of 2 mM DTPA solution to a final concentration of 500 µM. Each mixture was incubated for 5 min and purified by a centrifuged column procedure using Sephadex G-50 Fine, equilibrated and eluted with PBS to provide a solution of In-111-labeled mAbs. The radiochemical purity was >95% determined by size-exclusion high performance liquid chromatography (HPLC) (SE-HPLC) and TLC (online supplementary figure S1 ).
7
SULFO-TAG Labeling of Protein A/G
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After reconstitution in bicarbonate buffer (50 mM, pH 8.0), a solution of Recombinant Protein A/G (Thermo Scientific, cat # 77677) was prepared at a concentration at 2 mg/mL. MSD-Gold SULFO-TAG NHS-Ester (MSD, cat # R91AO-2) was suspended in dry DMF/water mixture, added promptly to the protein solution at a molar input ratio of about 12:1 (SULFO-TAG to Protein A/G), and allowed to react at ambient temperature in a sealed polypropylene vessel with gentle inversion to allow mixing. After about 2 h, the reaction was quenched by adding a small volume of amine-containing buffer, followed by gentle mixing for an additional 20 min. The SULFO-TAG labelled Protein A/G was purified by size exclusion chromatography using a HiPrep™ 26/40 column packed with Sephadex G-50 (fine) (GE, cat # 17-0042-01) and a mobile phase consisting of PBS supplemented with 5% sucrose and 0.05% Tween-20, pH 7.4. Detailed characterization of the purified conjugated product included concentration measurement by BCA Protein Assay, purity assessment by analytical HPLC-SEC and SULFO-TAG label molar incorporation ratio determination by Abs measurement at 455 nm. The conjugated protein was diluted into PBS containing 0.2% BSA buffer and stored in 0.1 mL aliquots in tightly sealed polypropylene vials at − 80 °C.
8
Purification of Bikaverin from Crude Extract
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Two open column chromatographic systems were tested in the present study to purify the bikaverin present in the crude extract. In the first, the concentrated crude extract resuspended in 0.5 mL chloroform was transferred to a glass column (18 cm height, 2.5 cm length, 0.8 cm width) filled with silica gel type H (20–80 μm particle size; code 815330.05 – Sigma), which was percolated with an eluting system consisted of a chloroform:methanol:acetic acid mixture (94:1:5, v/v/v), as described by Giordano (1999). Five mL fractions were collected for further analysis (Section 4.4.2). In the second case, the concentrated crude extract resuspended in 0.5 mL methanol was transferred to a glass column (29.5 cm height, 3.5 cm diameter) packed with Sephadex ™ – G-50 fine (70–230 mesh; GE Healthcare), which was percolated with methanol. Eight mL-fractions were collected for further analysis (Section 2.4.2 ).
9
Fluorescent Histone Octamer Reconstitution
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Amino acid substitutions in Xenopus laevis histone H3 (C110A, Q125C) and histone H2B (K113C) were introduced in the polycistronic plasmid pET29a‐YS14 by site‐directed mutagenesis. Recombinant histone octamers were expressed in E. coli and purified under native conditions as described (Shim et al, 2012 ) except that following NiNTA purification the peak histone‐containing fractions were incubated with a 40‐fold molar excess of tetramethylrhodamine‐5‐maleimide (Sigma‐Aldrich) overnight at 4°C. Unreacted dye was quenched with DTT and separated using Sephadex G50 Fine (GE Healthcare Life Sciences). Histone octamers were purified over a Superdex 200 column (GE Healthcare Life Sciences) and were then mixed with 1 μg of biotinylated pPlat or pPlat‐601 at a molar ratio of ~80 octamer:1 DNA in 10 mM Tris pH 7.6, 2 M NaCl, 1 mM EDTA, and incubated for 30 min at 4°C. Histone octamers were deposited on DNA by stepwise dilution at 4°C to reduce of NaCl concentration at 4°C (1 h at 1 M, 1 h at 0.8 M, 1 h at 0.67 M, 1 h at 0.2 M, overnight at 0.1 M). Reconstituted nucleosomal DNA was stored at 4°C.
10
Biochemical Reagents for Cell Culture
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TRIS, DMSO, Na2HPO4 and sodium acetate were from Merck (Darmstadt, Germany). EDTA was from Fluka (Buchs, Switzerland). Acrylamide, Commassie Brilliant Blue G-250 and Triton X-100 were from BDH (Poole, UK). RPMI 1640, streptomycin, penicillin, phorbol 12-myristate 13-acetate (PMA), Hepes, Brij-35, Silver nitrate, alkaline phosphatase-conjugated antibodies and gelatine were purchased from Sigma (St Louis, MO, USA). Gelatine-Sepharose, Q-Sepharose and Sephadex G-50 (fine) were from GE-Healthcare (Uppsala, Sweden). DC Protein Assay and unlabelled molecular weight standards were from BioRad (Richmond, CA, USA). Magic Marker molecular weight standards were from Invitrogen (Carlsbad, CA, USA). Western Blotting Luminol reagent and HRP-conjugated donkey anti-goat secondary antibody were from Sancta Cruz (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit secondary antibody was from Southern Biotech (Birmingham, AL, USA). Foetal bovine serum was from Biochrom AG (Berlin, Germany). Human MT1-MMP/MMP-14 (catalytic domain), TLN and PLN were from Calbiochem (San Diego, CA, USA) and Aureolysin was from BioCentrum Ltd (Kraków, Poland). McaPLGL(Dpa)AR-NH2 (ES001) and McaRPPGFSAFK(Dnp)-OH (ES005) were from R&D Systems (Minneapolis, MN, USA).
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