Shearing Technologies

The most important steps for a successful ChIP include both cell fixation and lysis, and chromatin shearing. Diagenode’s Bioruptor uses state-of-the-art ultrasound technology to give the highest chromatin quality for high IP efficiency and sensitivity for ChIP experiments with gentle yet highly effective shearing forces. Additionally, the Bioruptor provides a precisely controlled temperature environment that preserves chromatin from heat degradation such that protein-DNA complexes are well-preserved for sensitive, unbiased, and accurate ChIP.

Successful chromatin shearing from K562 cells using the Bioruptor Pico and the True MicroChIP Kit (Diagenode). Chromatin from K562 was sheared using the Bioruptor Pico for 5 cycles (30’’ ON/30’’ OFF). Chromatin was decrosslinked and purified accordingly to the protocol for chromatin shearing analysis and fragment size was assessed using the Fragment Analyzer (Agilent).

Chromatin immunoprecipitation analysis from K562 cells using control H3K27me3 and H3K9me3 antibodies. ChIP was performed on 25,000 cells using control H3K27me3 and H3K9me3 and negative IgG control antibodies following the True MicroChip protocol (Diagenode). Quantitative PCR was performed with the positive control TSH2B promoter and the negative control EIF2A primer sets. The recovery, expressed as a percent of input, is shown.

DNA shearing

Next Generation Sequencing (NGS) has revolutionized genomics and biology. One of the most critical aspects of optimal library preparation is the quality of the DNA to be sequenced. The DNA must first be effectively and consistently sheared into the appropriate fragment size (depending on the sequencing platform) to enable sensitive and reliable NGS results. The Bioruptor provides superior sample yields, fragment size, and consistency, which are essential for Next Generation Sequencing workflows.

High reproducibility with Bioruptor Pico Image shows peak electropherogram view (left), virtual gel view (center) and shearing conditions (right).

RNA shearing

RNA sequencing is a highly accurate and sensitive method to obtain unprecedented information about the transcriptome. The RNA must be fragmented to an appropriate size for sequencing prior to reverse transcription. The Bioruptor provides unbiased RNA shearing for best cDNA synthesis and ensures high quality Next Generation Sequencing.

Programmable RNA size distribution and excellent reproducibility with Bioruptor Pico. The various panels show different RNA size distributions of sheared total RNA produced by varying the duration of sonication on the Bioruptor. Panel A shows duplicate profiles produced after 5 (lanes 2-3), 10 (lanes 4-5) and 15 minutes (lanes 6-7) (30 sec on/off) of sonication. Lane 1 shows the unfragmented total RNA (starting material). Panel B and C compare the RNA size distributions of sheared total RNA from 2 different experiments. All samples were analysed on Biorad Experion using Eukaryote Total RNA HighSens chip.

Excellent reproducibility achieved with the Megaruptor 3 The Megaruptor 3 provides excellent shearing results for HiFi and microbial multiplexing libraries. Test shears of E.coli K12 are shown. The amount of gDNA input was 0.5 μg in a volume of 100 μl. Fragments lengths generated to 10 kb. Femtopulse analysis shows all 8 channels sheared within a variance of only 9%. Data generated by Pacific Biosciences.* *PacBio® and Pacific Biosciences are registered trademarks of Pacific Biosciences.

Various biochemical and analytical techniques require the extraction of protein from tissues or mammalian, yeast and bacterial cells. Obtaining high quality and yields of proteins is important for further downstream protein characterization such as in PAGE, western blotting, mass spectrometry or protein purification. The efficient disruption and homogenization of tissues and cultured cells obtained in just one step using the Bioruptor delivers high quality protein.
The PreOmics iST sample preparation of proteins combined with the Bioruptor Pico enhances mass spectrometric analyses. PreOmics shows that sample preparation of Baker’s yeast (Saccharomyces cerevisiae) with the iST Kit combined with the Bioruptor Pico outperforms the workflow with the heat treatment alone. Thus, the sonication-based cell lysis aids in proteome-wide discovery by greatly enhancing the number of peptide and protein identifications.

The formation of amyloid-like aggregates (amyloid fibrils) disturbs normal proteome function and contributes to toxicity in neurodegenerative disease.To study the properties of amyloid formation and cellular propagation, sonication is typically used for both preparing seeds by breaking apart amyloid fibers for new aggregate growth and stimulating the formation of amyloid aggregates. The Bioruptor provides a number of benefits in standardizing and creating representative seeding material with its closed system and controlled sonication and temperature.

EM images showing PFFs before and after sonication in the Bioruptor Plus after 10 cycles. See “Standardizing seeding experiments using the Bioruptor® for the understanding of the neuronal alpha-synuclein pathology” from Kelvin Luk, Ph.D., from the University of Pennsylvania to read more and see similar results using the Bioruptor Pico.

Various biochemical and analytical techniques require the extraction of protein from tissues or mammalian, yeast and bacterial cells. Obtaining high quality and yields of proteins is important for further downstream protein characterization such as in PAGE, western blotting, mass spectrometry or protein purification. The efficient disruption and homogenization of tissues and cultured cells obtained in just one step using Diagenode’s Bioruptor delivers high quality protein.

Simultaneous extraction of cytoplasmic, nuclear and chromatic proteins

Protein staining of cell lysates (containing both chromatin and soluble proteins) obtained with the protocol developed by Lauriane Fritsch from the laboratory of Slimane AIT-SI-ALI. A. Ponceau S Staining Western Blot of Hep G2 cells lysates obtained usting the Bioruptor. The rectangle indicates the location of histones. B. Western Blot of Hep G2 cells transfected and cultivated for 72h with siRNA against hMOF, a histone h4 K16-specific acetyl transferase. Both soluble (hMOf and B-actine) and chromatin (Histone 3 and 4) proteins are obtained on the same cell extract.

Most reliable and reproducible method to prepare lysates from C. elegans

Prasad Kasturi and F Ulrich Hartl at the Max Plank Institute for Biochemistry are using the Bioruptor for their research using C. elegans. SILAC quantitative proteomics showing the high reproducibility of proteome analysis in multiple replicates of worm samples of different ages (adopted from Walther DM and Kasturi P et al., Cell. 2015).

Read more about our protein extraction kit

Read more application notes for protein extraction:
Tissue and cell extraction
Western blot

Sonication is one of the most common methods employed for liposome preparation. The Bioruptor provides high throughput and reproducibility and eliminates the need for direct contact to prevent sample contamination. The precise temperature control preserves the lipids from damage through overheating or oxidation.

Size reduction of multilamellar vesicle

Peter Stone and Yvonne Perrie from Aston University and University of Strathclyde process MLV generated using the thin film lipid hydration method with the Bioruptor. The 100 µl samples are sonicated 15 minutes at 45°C in the Bioruptor Plus with high power for rapid small scale production of bilayer -loaded liposomes.

Deparaffinization of FFPE samples is typically performed using a non-polar solvent, such as xylene, or a mineral oil-based method which can be time consuming and messy. Using Diagenode’s Bioruptor is a superior method for removing the paraffin and rehydrating FFPE tissues in just one solvent-free step followed by a mild crosslink reversal to preserve DNA and RNA integrity.

Efficient deparaffinization of FFPE sections by sonication with Bioruptor 10 μm sections were sonicated for 3 cycles (30 sec ON/OFF at RT) with the Bioruptor Pico. The paraffin has been emulsified and completely dissociated from the tissue section.