Protein was prepared from 12 × 10 6 spermatozoa which were separated from round cells as for RNA extraction, and then lysed on ice for 30 min in 100 µL of 0.5% SDS, 50 mM Tris.HCl, pH 8.0, 5 mM EDTA, 50 mM DTT, 2× complete proteinase inhibitor cocktail (Roche), followed by 30 s of sonication at high setting in a Bioruptor Standard (Diagenode). The effectiveness of the round cell lysis is illustrated by the absence of a signal for Emerin, despite the strong signal in whole testis (Supplementary Fig. 4). Emerin is strongly detected in testicular somatic cells, but we did not detect it by IF in spermatids or spermatozoa. For testis lysates, 60 mg of tissue was dissociated in 300 mL of lysis buffer (1 M Tris.HCl, pH 7.5, 5 M NaCl, 0.1 mM EGTA, 10% Triton) using ceramic beads in a Magnalyser (Roche), followed by 30 min on ice and 3× 30 s of sonication at high setting in a Bioruptor Standard (Diagenode). For migration, lysate from approximately 2 million spermatozoa or 40 µg of testis protein was mixed with 2× Laemmli sample loading buffer (Sigma Aldrich) with 5% β-mercaptoethanol, heated to 95°C for 5 min and subjected to SDS-PAGE. Proteins were blotted on to nitrocellulose with a pore size of 0.45 µm (Emerin) or 0.2 µm (BAF, BAF-L).
Bioruptor standard
Manufactured by Diagenode
Sourced in Belgium
The Bioruptor Standard is a laboratory device designed for efficient and reproducible sonication of samples. It utilizes ultrasonic waves to disrupt cellular structures and extract biomolecules, such as DNA and proteins, from various biological samples. The Bioruptor Standard allows for controlled and consistent fragmentation of samples, enabling downstream applications like chromatin immunoprecipitation (ChIP) and next-generation sequencing (NGS).
Automatically generated - may contain errors
Lab products found in correlation
Formaldehyde, by Merck Group (3 mentions)
Purvalanol a, by Merck Group (1 mentions)
Roscovitine, by Merck Group (1 mentions)
Rabbit igg sc 2027, by Santa Cruz Biotechnology (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Ab110641, by Abcam (1 mentions)
Flag m2 agarose, by Merck Group (1 mentions)
Amersham protran, by Merck Group (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Protease, by Roche (1 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
4 thio uracil 4su, by Merck Group (1 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Ionxpress plus gdna fragment library preparation rev c 0, by Thermo Fisher Scientific (1 mentions)
Ha peptide, by Merck Group (1 mentions)
16 protocols using bioruptor standard
1
Western Blot of Spermatozoa Proteins
2
Circadian Rhythm Modulation Proteomics
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Confluent U2OS cell cultures grown in 150-mm dishes (2 × 107) were treated with circadian period–altering compounds (longdaysin [cat. no. SML0127; Sigma-Aldrich], purvalanol A [cat. no. P4484; Sigma-Aldrich], roscovitine [cat. no. 557360; Calbiochem] and SP600125 [cat. no. S5567; Sigma-Aldrich]) to a final concentration of 10 μM dissolved in DMSO (100 μl of DMSO; <0.5% of the total culture volume) or an equivalent amount of DMSO (vehicle only). After 48-h incubation with the compounds or DMSO, the cells were harvested by trypsinization, washed twice in PBS, and were lysed in 500 μl lysis buffer containing 50 mM Hepes (pH 8.5), 8M urea, 1% NP-40, protease and phosphatase inhibitors, and benzonase nuclease. Then mild sonication was applied for 15 min (30 s on, 30 s off; medium power) using a Bioruptor Standard (Diagenode) instrument and lysates were centrifuged at 16,000g for 20 min at 4°C. Supernatants were carefully separated and transferred into new microcentrifuge tubes. Protein precipitation was performed with 1:6 volume of prechilled (−20°C) acetone overnight at 4°C. After overnight incubation, the lysates were centrifuged at 14,000g for 15 min at 4°C. Supernatants were discarded without disturbing pellets, and the pellets were air-dried for 2–3 min to remove residual acetone. Then the pellets were dissolved in 500 μl 100 mM triethylammonium bicarbonate (TEAB) buffer.
3
ChIP-seq Assay for Histone Marks
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ChIP assays were performed as described previously with minor modifications 49 (link). Briefly, cells were cross-linked at a concentration of 2 × 106 cells/mL with 1% formaldehyde for 6 min at RT. Cross-linking was stopped with 0.125 M glycine. Chromatin sonication was performed with a Diagenode Bioruptor Standard with cooling device at 4°C for 10 min with 30 s pulse/pause cycles or with a Covaris M220 Focused-ultrasonicator device for 3 min. Ten microliters Dynabeads Protein A (10002D; Life Technologies) were incubated with either 2 μg rabbit anti-H3K9ac antibody (pAb-004–050; Diagenode), 2 μg rabbit anti-PU.1 antibody (T-21, sc-352; Santa Cruz), or 1 μg rabbit IgG (sc-2027; Santa Cruz) at 4°C and 40 rpm. Sheared chromatin from 0.5 – 1 × 106 cells was added to the preincubated beads and incubated at 4°C and 40 rpm overnight. Bound chromatin complexes were magnetically selected (Dynal) and washed with RIPA and TE buffer. DNA was purified using NucleoSpin Gel and PCR Clean-up kit (Macherey & Nagel) or QIAquick PCR purification kit (Qiagen) according to the provided protocol and used for qPCR. Enrichment was calculated as percentage of input. MacVector software package (MacVector Inc., Cary, NC, USA) was used for primer design. Alternatively, published primers were used as indicated. Primer sequences are provided in Supporting Information Table 2 and 3.
4
ChIP-seq analysis of Pax5 and Brg1
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Pro-B cells were short-term cultured on OP9 cells in IL-7–containing IMDM (Nutt et al., 1997 (link)) followed by crosslinking with 1% formaldehyde (Sigma-Aldrich) for 10 min. Nuclei were prepared and lysed in the presence of 0.25% SDS, followed by sonication of the chromatin with the Bioruptor Standard (Diagenode). IP was performed with an anti-Pax5 paired domain antibody (Adams et al., 1992 (link)), and the precipitated DNA (1–2 ng) was used for library preparation and subsequent Illumina deep sequencing (Table S7 ). For Brg1 ChIP analysis, in vitro cultured pro-B cells were sequentially crosslinked at room temperature with 1.5 mM ethylene glycol bis(succinimidylsuccinate) for 15 min and then with 1% formaldehyde for 10 min, followed by ChIP analysis with an anti-Brg1 antibody (ab110641; Abcam), as described (Wang et al., 2020 (link)).
5
Protein Extraction and Western Blot
6
ChIP Assay with Flag Agarose
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ChIP was performed as described previously (Jha and Strahl 2014 (link)) with some exceptions. Sonication for each sample was performed for 20 min with alternating on/off cycles of 30 sec using a Bioruptor Standard (Diagenode). Immunoprecipitation was performed overnight with 1 mg of clarified, sonicated extract and 20 µL of equilibrated Flag M2 agarose (Sigma). Primer sequences are listed in Supplemental Table 3, and analysis methodologies are described further in the Supplemental Material.
7
Western Blot Protein Extraction and Detection
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Cells were collected after two cold PBS washes by scraping in 2X SDS Lysis Buffer (4% SDS, 20% Glycerol, 120 mM Tris-Cl pH 6.8, 1x protease (Roche) and phosphatase inhibitors (Sigma-Aldrich)) on ice. Lysates were sonicated at medium-high intensity for 10 minutes in a Bioruptor Standard (Diagenode) placed at 4°C and subsequently boiled for 10 minutes at 90°C. Proteins were quantified using the DC Protein Assay (Bio-Rad), separated by SDS-PAGE and transferred to 0.2 μm pore Nitrocellulose membrane (Amersham Protran; Sigma-Aldrich). Membranes blocked with 5% milk/PBST for 1 h at room temperature and probed with primary antibodies overnight at 4°C. These were detected with appropriate secondary antibodies conjugated to HRP and visualized by ECL-Plus (GE Healthcare).
8
ChIP-qPCR for ERT2 Binding Assessment
9
Pax5 ChIP-seq in Pro-B Cells
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Pro-B cells were short-term cultured on OP9 cells in IL-7–containing IMDM (Nutt et al., 1997 (link)) followed by crosslinking with 1% formaldehyde (Sigma-Aldrich) for 10 min. Nuclei were prepared and lysed in the presence of 0.25% SDS, followed by sonication of the chromatin with the Bioruptor Standard (Diagenode). Immunoprecipitation was performed with an anti-Pax5 paired domain antibody (Adams et al., 1992 (link)), and the precipitated DNA (1–2 ng) was used for library preparation and subsequent Illumina deep sequencing (Table S8 ).
10
4sU-labeling and TT-seq in Yeast
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TT-seq experiment was performed as described33 (link) for two biological replicates. Briefly, 1.5 × 107S. cerevisiae cells were labeled with 2.5 mM of 4-thiouracil (4sU) (Sigma-Aldrich) for 10 min. Cells were harvested by centrifugation at 3000g for 2 min. Total RNA was extracted with hot acid phenol as described above. RNAs were sonicated to generate fragments of <1.5 kbp using Bioruptor Standard (Diagenode). 4sU-labeled RNA was purified from 150 μg total fragmented RNA. Labeled RNA was separated with streptavidin beads (Thermo Fisher Scientific). Strand-specific library preparation for labeled RNA was performed as described53 (link) and as in RNA-seq section above. After library construction, size of the library was determined by fragment analyzer and the concentrations were determined by Qubit 4.0 fluorometer (Invitrogen). Sequencing of all samples was carried out on an Illumina NextSeq 500 with a read length of 150 (paired-end).
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