Genetic Engineering & Biotechnology News

NOV1 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Detection of a Low-Abundant Epigenetic Protein Post- Translational Modifi cation using a Duolink ® fl owPLA Detection Kit Epigenetics (heritable changes in gene expression that occur without alteration in DNA sequence) is an important factor in cancer development and works by altering the regulation of genes involved in cell growth, survival, or diff erentiation through histone modifi cations. Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that catalyzes the trimethlyation of lysine 27 of histone H3. This modifi cation leads to transcriptional repression and is prevalent in many types of human cancers, such as liver, breast, and prostate cancer. Prior to the advent of Duolink ® PLA technology, the interaction between EZH2 and histone H3 at K27 would have been diffi cult to detect by microscopy due to the transient nature of the interaction. To query this interaction in large cell populations by traditional fl ow cytometry would be impossible as fl ow cytometry can only measure co-expression levels, not protein interactions, and the low abundant nature of an epigenetic marker would make it challenging to detect. To showcase the power of Duolink ® PLA technology in studying protein interactions, post-translational modifi cations, and low- abundant targets via fl ow cytometry, the EZH2/ H3K27me3 interaction was queried. DU145 human prostate cancer cells were grown on a chambered slide, fi xed, permeabilized, and blocked for analysis by microscopy. Alternatively, the DU145 cells were detached, then fi xed, permeabilized, blocked in suspension, and aliquoted into 96-well plates for analysis by fl ow cytometry. The Duolink ® assay was performed using rabbit anti-H3K27me3 (SAB800015) and mouse anti-EZH2 (415M-156) primary antibodies, anti-rabbit PLUS and anti- mouse MINUS PLA probes, and the Duolink ® fl owPLA Detection Kit – FarRed, according to the Duolink ® PLA Flow Cytometry Protocol. Technical negative controls included incubation with each primary antibody separately and no primary antibody. A schematic of this Duolink ® PLA reaction is shown in Figure 3D. A few PLA signals, as seen as red punctate spots, were detected in the nuclei of DU145 cells by fl uorescence microscopy (Figure 3A) , showing the low abundance of the EZH2/ H3K27me3 interaction. However, detecting this interaction by fl ow cytometry was diffi cult, as indicated by the slight shift in the Duolink ® PLA signal intensity of the orange peak compared to the green peak in Figure 3B, presumably due to the low abundance of this interaction. By extending the Duolink ® PLA amplifi cation time from the standard 100 minutes to overnight, enhanced detection was achieved (Figure 3B, pink peak). Minimal PLA signal was detected in the single primary antibody negative controls. Of note, extending the amplifi cation time using the original Duolink ® PLA detection reagents resulted in higher background because the fl uorescent-labeled detection oligonucleotides are supplied in the amplifi cation buff er. In the Duolink ® fl owPLA Detection Kits, the fl uorescent- labeled detection oligonucleotides are supplied in a separate detection buff er, allowing more fl exibility in the amplifi cation and detection times for the user to obtain optimal results. Combining Duolink ® PLA technology with imaging fl ow cytometry allows localization of proteins or protein events (interactions or modifi cations) in large cell populations. We next analyzed the EZH2/H3K27me3 interaction using the Amnis ImageStream X ® (Figure 3C). Discrete Duolink ® PLA signals were detected in cells when the amplifi cation incubation was the standard 100 minutes. However, the extension of the amplifi cation time to overnight resulted in coalesced signals. Therefore, extended amplifi cation time for low- abundant targets can cause coalescence of the individual PLA signals and may be unnecessary for imaging fl ow cytometry due to the exquisite sensitivity of the optics of the system. Figure 3. Increased amplifi cation time during Duolink ® PLA can aid in the detection of low- abundant protein targets by fl ow cytometry. Duolink ® PLA was performed to detect the trimethylation of lysine 27 on histone 3 (H3K27me3) mediated by EZH2. A) Few PLA signals (red) in the nuclei (blue) of DU145 cells were detected by fl uorescence microscopy after 100 min amplifi cation. FITC- Phalloidin-stained actin (green) was used as a counterstain. B) Extended amplifi cation times enhanced the detection of EZH2-H3K27me3 interactions by A B C D No primary Ab EZH2/H3K27me3 PLA, 100 min amplification EZH2/H3K27me3 PLA, O/N amplification Anti-mouse MINUS Anti-rabbit PLUS Mouse anti-EZH2 Rabbit anti-H3K27me3 EZH2 H3 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. conventional fl ow cytometry. C) Analysis of the EZH2-H3K27me3 interactions by imaging fl ow cytometry provided localization, but extended amplifi cation causes coalescence of the PLA signals. D) Schematic of the Duolink PLA reaction. In summary, the data presented here demonstrate the power of Duolink ® PLA technology as a tool that can enable the use of fl ow cytometry to query protein-protein interactions, post-translational modifi cations, and low- abundant protein events. The fl exibility of the technology allows the use of a multitude of fl ow cytometry instrumentation with the appropriate fi lter sets. The ability to use fl ow cytometry as a read-out for these types of interactions provides an exciting new avenue for protein detection. For more information on the Duolink ® fl owPLA Detection Kits, visit owpla Key Sample ID • EZH2/H3K27me3 PLA, O/N amplifi cation • EZH2/H3K27me3 PLA, 100 min amplifi cation • Anti-EZH2 antibody only control, O/N amplifi cation • Anti- H3K27me3 antibody only control, O/N amplifi cation • No primary antibodies control, 0/N amplifi cation Figure 3

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