The nucleosomes were reconstituted by the salt dialysis method42 (link),43 (link), with the histone octamer and a DNA fragment. The reconstituted nucleosomes were further purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad).
Model 491 prep cell
Manufactured by Bio-Rad
Sourced in United States
The Model 491 Prep Cell is a laboratory instrument designed for preparative electrophoresis. It is used to separate and purify macromolecules, such as proteins and nucleic acids, based on their size and charge.
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Sw40 rotor, by Beckman Coulter (1 mentions)
Caps buffer, by Merck Group (1 mentions)
Model 422 electro eluter, by Bio-Rad (1 mentions)
Novex sharp pre stained protein standard, by Thermo Fisher Scientific (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Cho k1, by American Type Culture Collection (1 mentions)
Atcc ccl 61, by American Type Culture Collection (1 mentions)
Puc57 vector, by GenScript (1 mentions)
Ultrafree 15 centrifugal filters, by Merck Group (1 mentions)
23 protocols using model 491 prep cell
1
Nucleosome Reconstitution and Purification
2
DNA Nucleosome Reconstitution Workflow
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DNA fragments for nucleosome reconstitution were amplified by PCR and purified by native polyacrylamide gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad). The SHL5e DNA fragment was amplified in Escherichia coli cells and was purified for the cryo-EM analysis. The DNA sequences used in this study are derived from the Widom 601 sequence17 (link) and native human genome sequence. The native human genomic region (GRCh37/hg19 chr4:123,459,211–123,459,355) was selected based on the Capture MNase-seq data (Supplementary Fig. 1d ). The DNA sequence of the nucleosomes used in this work are presented in full in Supplementary Table 6 .
3
Recombinant Histone H1.2 Nucleosome Reconstitution
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The human linker histone H1.2 was produced in and purified from bacterial cells as described previously60 (link). Mononucleosomes were reconstituted with 145 bp or 193 bp of the Widom 601 sequence61 (link),62 (link) by the salt-dialysis method as described previously63 (link). The resulting nucleosomes were purified by native PAGE with the use of a Prep Cell model 491 apparatus (Bio-Rad).
4
Preparation and Purification of Marine Peptides
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Chemicals used to prepare solutions and buffers were of high-quality reagent grade from Sigma-Aldrich (Madrid, Spain), unless stated otherwise. PTHrP(1–34) and PTHrP(7–34) were synthetized by Genemed Synthesis, Inc (San Francisco, CA) and were based on the puffer fish, Takifugu rubripes, PTHrPA sequence, which is highly conserved across vertebrates1 (link). STC1A was extracted from European sea bass CS and purified by SDS-PAGE using a Prep Cell Model 491 (Bio-Rad)8 (link). The anti-sea bream (Sparus aurata) STC1A serum was the one from Gregorio et al.40 (link).
5
CENP-A, H3.1, or H3.1CATD Nucleosome Purification
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The purified CENP-A, H3.1, or H3.1CATD nucleosomes (463 nM) were incubated with the CENP-B DBD (2.6–3.5 μM) for 20 min at 37ºC. The nucleosomes complexed with the CENP-B DBD were separated from the free nucleosomes and the CENP-B DBD by preparative non-denaturing polyacrylamide gel electrophoresis (Prep Cell Model 491: Bio-Rad). The purified nucleosome-CENP-B DBD complexes were concentrated and stored on ice.
6
Preparative SDS-PAGE Purification of Recombinant Profilin
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Using preparative SDS-PAGE (prep cell model 491) from Bio-Rad (Hercules, CA, USA), the recombinant profilin, which was expressed and isolated under native conditions, was separated from further contaminants. The pooled and washed samples from the elution (above) were used in a ratio of 1:2 in a 2-fold-reducing, or 1:5 in a 5-fold-reducing sample buffer and then heated for approx. 5 min at 95 °C. For the preparative SDS-PAGE, an 11% separation gel and a 4% collection gel were used.
The column was constructed and filled with the separating gel. Then the gel was covered with water-saturated n-butanol and left to polymerize overnight. The gel was washed with Milli-Q water, and the collection gel was installed onto the separating gel. The column with the now polymerized gel was installed in the prep cell, which was filled with running buffer. The sample was applied and separated in the gel at 150 V for 80 min. The voltage was increased to 250 V for the remaining electrophoresis time.
After the bromophenol peak became visible, 95 fractions of 6 mL each were collected. The collected fractions were then investigated for the recombinant profilin.
The column was constructed and filled with the separating gel. Then the gel was covered with water-saturated n-butanol and left to polymerize overnight. The gel was washed with Milli-Q water, and the collection gel was installed onto the separating gel. The column with the now polymerized gel was installed in the prep cell, which was filled with running buffer. The sample was applied and separated in the gel at 150 V for 80 min. The voltage was increased to 250 V for the remaining electrophoresis time.
After the bromophenol peak became visible, 95 fractions of 6 mL each were collected. The collected fractions were then investigated for the recombinant profilin.
7
Reconstitution and Purification of Nucleosomes
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The CENP-A, H3.1 and H3.1CATD nucleosomes were reconstituted with the 166 base-pair satellite DNA fragment by the salt dialysis method, as described previously (15 (link),46 (link),48 (link)). The reconstituted nucleosomes were purified by preparative non-denaturing polyacrylamide gel electrophoresis (Prep Cell Model 491: Bio-Rad).
8
Reconstitution and Purification of Specialized Nucleosomes
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Canonical NCP, NCPCENP-A, and NCPapd were reconstituted by the salt dialysis method, with the 145-base pair Widom 601 DNA and a histone octamer containing H2A-H2B-H3.1-H4, H2A-H2B-CENP-A-H4, or the H2Aapd-H2Bapd-H3.1-H4 (Tachiwana et al, 2010 (link)). After dialysis, the NCPs were purified with a Prep Cell Model 491 apparatus (Bio-Rad), using a native polyacrylamide gel. The NCPs were eluted in TCS buffer, which contains 20 mM Tris–HCl (pH 7.5) and 1 mM dithiothreitol.
9
Preparative Gel Electrophoresis for Serum Albumin
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Denaturing preparative gel electrophoresis was performed with PrepCell Model 491 instrument (BioRad, CA) to separate albumin from other serum proteins. For separation, three different gel formats were tested; (1) a stepwise gel that was composed of 1.5 cm 15%, 3 cm 12% and 1.5 cm 10% polyacrylamide gel layers and (2) an 8% – 15% gradient gel and (3) a 12% polyacrylamide gel. Total length of each separating gel was kept at 6 cm. Each separating gel was also topped with 1 cm, 4% stacking gel. The bottom of PrepCell instrument was sealed with a dialysis membrane that had a molecular weight cut-off limit of 5 kDa. For each run, 15 mg protein from an lgG-depleted serum sample was mixed with 6X loading dye and incubated at 62°C for 20 minutes. After incubation and a brief cooling on ice, the sample was loaded onto a preparative gel and run at 25 mA for 20 minutes. The current was then raised to 45 mA and the gel was run with this constant current till the experiment was completed. The collection of the fractions was started right after the front blue dye eluted off the column. Three hundred fractions (0.5 mL/fraction) were collected using an automated fraction collector (BioRad, CA) to which a peristaltic pump (EP-1 Econo, BioRad, CA) was attached. The pump was set to a speed of 0.5 mL/min. Three independent experiments were carried out to assess reproducibility.
10
2'-F RNA Aptamer Synthesis and Characterization
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The aptamers used in this study were previously discovered using SELEX (Huang et al, 2007 (link), 2017 (link)). A 2′-F–modified RNA aptamer was prepared by transcribing the corresponding DNA template. To synthesize 2′-F RNA aptamer from its DNA template, we incorporated non-canonical 2′-F-NTP (i.e., “N” in the “NTP” stands for adenosine [citidine, uracil] triphosphate ATP, CTP, and UTP), using a mutant T7 RNA polymerase. However, all G (guanosine) positions contained the regular, unmodified G (Huang et al, 2007 (link), 2017 (link)). A transcribed RNA was purified using a Bio-Rad Prep Cell (model 491), a PAGE-based, continuous elution apparatus, coupled with a Q column (Huang et al, 2007 (link), 2013 (link)). A purified RNA aptamer was tested for its biological activity against its target using whole-cell recording (see Fig 1 and legend), and the procedure of whole-cell recording was previously described (Huang et al, 2013 (link)).
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