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This 5X binding buffers where experiment participates in?

This 5X binding buffers where experiment participates in?


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I saw 5X bingind buffer maded by;

  • 100mM HEPES, pH 7.6
  • 5mM EDTA
  • 50mM ammonium sulfate
  • 5mM DTT
  • 1% Tween-20
  • 150mM potassium chloride

which reaction helps this buffer? I don't consider to this buffer participates in which experiment by name. Although this buffer have another name but they writes just '5X bonding buffer'…


Agarose

Agarose is a polysaccharide, generally extracted from certain red seaweed. [1] It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. [2] Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin. [3]

Agarose is frequently used in molecular biology for the separation of large molecules, especially DNA, by electrophoresis. Slabs of agarose gels (usually 0.7 - 2%) for electrophoresis are readily prepared by pouring the warm, liquid solution into a mold. A wide range of different agaroses of varying molecular weights and properties are commercially available for this purpose. Agarose may also be formed into beads and used in a number of chromatographic methods for protein purification.


TBE Uses

TBE buffer is particularly useful for the separation of smaller DNA fragments (MW < 1000), such as small products of restriction enzyme digests. TBE has a greater buffering capacity and will give sharper resolution than TAE buffer. TAE (Tris-acetate-EDTA) buffer is a solution made up of Tris base, acetic acid, and EDTA.

TBE is generally more expensive than TAE and inhibits DNA ligase, which may cause problems if subsequent DNA purification and ligation steps are intended. With the three simple steps that follow, learn how to make TBE buffer. It shouldn't take more than about 30 minutes to create.


There are several recipes to prepare PBS solution. The essential solution contains water, sodium hydrogen phosphate, and sodium chloride. Some preparations contain potassium chloride and potassium dihydrogen phosphate. EDTA may also be added in cellular preparation to prevent clumping.

Phosphate-buffered saline is not ideal for use in solutions that contain divalent cations (Fe 2+ , Zn 2+ ) because precipitation may occur. However, some PBS solutions do contain calcium or magnesium. Also, keep in mind phosphate may inhibit enzymatic reactions. Be particularly aware of this potential disadvantage when working with DNA. While PBS is excellent for physiological science, be aware the phosphate in a PBS-buffered sample may precipitate if the sample is mixed with ethanol.

A typical chemical composition of 1X PBS has a final concentration of 10 mM PO4 3− , 137 mM NaCl, and 2.7 mM KCl. Here's the final concentration of reagents in the solution:

Salt Concentration (mmol/L) Concentration (g/L)
NaCl 137 8.0
KCl 2.7 0.2
Na 2HPO 4 10 1.42
KH 2PO 4 1.8 0.24

Acknowledgements

We thank S. Berget (Baylor College of Medicine) for the CD44 v4-v5 minigene, T. Cooper (Baylor College of Medicine) for YB-1 plasmids, J. Tazi (Institut de Génétique Moléculaire de Montpellier) for plasmid pET14b-hSC35, D. Barford (Chester Beatty Laboratories) for recombinant PP2Cα protein, G. Dreyfuss (University of Pennsylvania School of Medicine) for the hnRNP A1–specific 4B10 antibody, S. Dokudovskaya and C. Muchardt for useful comments on the manuscript, and E. Batsché for helpful advice on the qPCR experiments. This work was supported by US National Institutes of Health grant GM42699 to A.R.K.


4. Discussion

In this study, we have found that FUBP1 protein increases its binding to Nrf2 mRNA and is required for de novo Nrf2 protein translation under oxidative stress. Mechanistically, FUBP1 did not show cytoplasmic translocation, but is present in 40/43S ribosomal fraction and shows a H2O2 dose dependent increase in total ribosomal fractions. Interestingly, FUBP1 interacts with eIF3η specifically, suggesting its role in attaching 43S pre-initiation complex to Nrf2 mRNA for translation initiation. Our data have revealed a novel mechanism of oxidative stress induced de novo Nrf2 protein translation.

FUBP1 protein was first discovered as a co-activator for maximizing c-myc transcription due to its binding to F ar U p s tream E lement (FUSE), in an AT-rich region 𢄡.5 kb upstream of the c-Myc promoter [24]. The core of FUSE in c-myc gene is 5′-TATATTCCCTCGGGATTTTTTATTTTGTG-3’ [24]. Through binding to FUSE, FUBP1 coordinates with additional cis-elements and their corresponding transcription factors to maximize the rate of transcription of the c-Myc gene. Aberrant expression of FUBP1 has been associated with various types of cancers, therefore emerging as a novel oncogene.

FUBP1 can bind to RNA at pyrimidine rich sequences and has been reported to bind several cellular mRNA species and viral RNA strands [24]. This protein contains four tandem K-homology (KH) motifs, similar to those in Heterogeneous Nuclear Ribonucleoprotein K (hnRNP-K) [25]. KH motifs mediate protein binding to single stranded DNA or RNA [26]. In fact, KH3 or KH4 of hnRNP is sufficient for binding to single stranded TTTT or ATTC sequence, respectively [27]. Nrf2 5′UTR contains one UUUU sequence and one AUUAC, and 7 patches of pyrimidine rich sequences, providing the potential binding sites for FUBP1. Importantly FUBP1 has been reported to play a role in IRES mediated translation of p27 Kip mRNA [24]. Our data showing FUBP1 binding to Nrf2 mRNA are consistent with a role of FUBP1 in IRES mediated Nrf2 protein translation under oxidative stress.

FUBP1 is also known as an RNA splicing factor. The observed nuclear localization of FUBP1 supports its potential role as a splicing factor. FUBP1 promotes or facilitates splicing of Duchenne Muscular Dystrophy (DMD) gene and MDM2 oncogene [28,29]. Splicing regulatory elements are present in the exons and introns either as an enhancers or silencers. FUBP1 can bind to an exonic splicing silencer of cardiac triadin gene to repress its alternative splicing [30]. Recently, NCBI and Ensembl Genomic databases indicate that Nrf2 gene encodes 8 or 14 transcripts respectively, suggesting that Nrf2 gene undergoes alternative splicing. The interplay between alternative splicing and protein translation remains to be studied.

FUBP1 joins La/SSB and EF1a in the list of proteins found to increase binding to Nrf2 mRNA when cells are experiencing oxidative stress. We did not find FUBP1 interaction with La/SSB or EF1a, suggesting that each of these proteins bind to a distinct area of Nrf2 mRNA. Additionally, FUBP1 appears to respond to oxidative stress in a manner different from La/SSB or EF1a. While EF1a binds to the G-quadruplex in Nrf2 5′UTR [13], La/SSB translocates from the nuclei to the cytoplasm upon oxidative stress [12]. Cytoplasmic translocation of FUBP1 was reported in early phase of Japanese Encephalitis Virus (JEV) or Enterovirus 71 (EV71) infection [31,32]. Unlike these viral proteins, Nrf2 protein translation via FUBP1 participation does not appear to involve its nuclear to cytoplasmic translocation. The fact that FUBP1 was detected in the cytosol and increased its presence in the ribosomal fractions suggests that cytosolic redistribution of FUBP1 to be associated with ribosomes is important for de novo Nrf2 protein translation.

The interaction between FUBP1 and eIF3η was enhanced upon H2O2 treatment ( Fig. 13 ). The initiation factor eIF3η is one of 13 subunits for 800 kD eIF3 complex that promotes assembly of the 43S pre-initiation complex and its association with the mRNA pre-occupied with eIF4F complex (eIF4E, eIF4G and eIF4A). With IRES mediated viral protein translation, involvement of eIF3 has been long established [33,34]. For cellular IRES, eIF3 has been shown to mediate 5′UTR dependent translation initiation of XIAP and c-Jun [[35], [36], [37]]. The physical interaction of FUBP1 with eIF3η discovered here supports a role of FUBP1 in promoting the attachment of 43S pre-initiation ribosomal complex to Nrf2 mRNA for translation initiation.

A plausible explanation for the observed increase of FUBP1 binding to Nrf2 mRNA is that FUBP1 undergoes posttranslational modifications upon oxidative stress. FUBP1 was reported to be ubiquitinated by p38/JTV-1 at the C-terminus, leading to its proteolysis [38]. Interestingly, FUBP1 can also be de-ubiquitinated by Ubiquitin-specific protease 22 (USP22), resulting in a reduction of FUBP1 binding to FUSE [39]. Posttranslational modifications, such as ubiquitination or sumoylation cause changes the molecular weights and/or charges of proteins, which can be detected by 2-dimensional electrophoresis. Lack of molecular weight change in 2-D Western blot argues against ubiquitination or sumoylation in FUBP1 due to H2O2 treatment. While 2-D electrophoresis showed a lowered PI of FUBP1 in cells treated with H2O2 treatment, antibodies detecting phosphorylation failed to indicate such posttranslational modification. Initial detection of FUBP1 using LC-MS/MS did not reveal phosphorylation or ubiquitination. Therefore, the signaling pathway leading to increased FUBP1 binding to Nrf2 mRNA remains to be determined.


INTRODUCTION

In various physiological and pathological events, such as embryonic development, inflammatory and immune responses, and cancer metastasis, cell migration responding to environmental cues is a pivotal mechanism (Bravo-Cordero et al., 2012 Solnica-Krezel and Sepich, 2012 Kolaczkowska and Kubes, 2013). The migration process requires the coordination of signaling pathways and the motility machinery (Insall, 2013), and the complex underlying molecular network remains to be fully elucidated.

Eukaryotic cell migration generally involves drastic cell shape changes driven by the rearrangement of cytoskeleton. In the crawling movement of cells, continuous reorganization and turnover of the actin cytoskeleton occur (Pollard and Borisy, 2003). At the cell front, rapid actin polymerization drives the extension of membrane protrusions such as lamellipodia and filopodia (Bisi et al., 2013). The new cellular protrusions adhere to the substratum through proteins that can engage the extracellular matrix to provide anchor points. For optimal migratory movement, a cell needs the contraction ability to drive the translocation of the trailing cell body this ability depends on the interaction between actin filaments and myosin (Cramer, 2013). The cell rear is detached from the original adhesion to allow the cell to advance a step. These events proceed in a cyclical manner and are spatially and temporally coordinated (Lauffenburger and Horwitz, 1996 Maruthamuthu et al., 2010).

Dynamic reorganization of the actin cytoskeleton is intricately controlled by a myriad of actin-binding proteins (ABPs Winder and Ayscough, 2005). Two forms of actin—globular monomeric G-actin and filamentous polymeric F-actin—coexist in cells in a dynamic equilibrium ATP–G-actin monomers add to the barbed end, and ADP–G-actin subunits dissociate from the pointed end of F-actin. The released ADP–G-actin monomers undergo nucleotide exchange to replenish the cellular ATP–G-actin pool for new rounds of actin filament assembly. Many ABPs have been identified and categorized into a few major families based on the actin-binding domains, including the actin-depolymerizing factor homology (ADF-H) domain, the gelsolin homology domain, the Wiskott–Aldrich syndrome protein (WASP) homology-2 (WH2) domain, the calponin homology (CH) domain, and the myosin motor domain (Paunola et al., 2002 McGough et al., 2003 Disanza et al., 2005 Friedberg and Rivero, 2010 Poukkula et al., 2011 Hartman and Spudich, 2012). ABPs can associate with G-actin and/or F-actin and regulate the actin cytoskeleton in different ways (Winder and Ayscough, 2005). For example, the Arp2/3 complex binds to actin and serves as a nucleation factor to promote branching of the filaments (Machesky et al., 1997 Machesky and Insall, 1998). The ADF/cofilin family members are disassembly factors with filament-severing activities (Prochniewicz et al., 2005). Other activities of ABPs include filament capping, debranching, monomer binding, bundling, and cross-linking (Winder and Ayscough, 2005). The repertoire of actin regulators is still expanding, and the cellular roles of many ABPs are still elusive.

Dictyostelium discoideum is a simple eukaryote that exhibits chemotactic migration in multiple stages of its life cycle. During growth, Dictyostelium amoebae migrate toward bacteria and consume them by phagocytosis as their food. Under nutrient depletion, Dictyostelium cells enter a developmental program in which single cells collectively move toward the cAMP signals released from designated central cells, forming aggregates that later undergo differentiation and morphogenesis to turn into multicellular structures (Kay, 2002 Weijer, 2009). With the many available molecular genetics tools and the haploid state ideal for genetic screening, Dictyostelium has been extensively exploited in studying cell migration and actin regulation (Egelhoff and Spudich, 1991 Noegel and Schleicher, 2000).

To uncover novel molecular players in the pathways underlying chemotactic cell migration, we previously performed a screen for Dictyostelium mutants defective in chemotactic responses to cAMP (Pang et al., 2010). In the present study, we analyzed a mutant collected in the screen and identified a novel actin-binding protein involved in modulating cell migration.


A typical recipe for making 1X TE buffer is:

TE buffer is also called as T10E1 Buffer, and read as "T ten E one buffer". To make a 100 ml solution of T10E1 Buffer, 1 ml of 1 M Tris base (pH 10–11) and 0.2 ml EDTA (0.5 M) are mixed and made up with double distilled water up to 100ml. Add microliter amounts of high molarity HCl to lower the pH to 8.

Based on nuclease studies from the 1980s, the pH is usually adjusted to 7.5 for RNA and 8.0 for DNA. [ citation needed ] The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA [ citation needed ] .

EDTA further inactivates DNase, by binding to metal cations required by this enzyme. [1]

Genomic and plasmid DNA can be stored in TE Buffer at 4 °C (39.2 °F) for short-term use, or -20 °C (-4 °F) to -80 °C (-112 °F) for long-term storage. Repeated freeze-thaw cycles should be avoided. [2]

The operation of the TE buffer is based on chelating metal cations such as Mg 2+ . The problem is that the PCR polymerase also requires Mg 2+ to function, so if the amount of EDTA is too high it can affect the PCR. There is a version of TE buffer with 10 times less amount of EDTA that is very frequently used for the forensic STR kits. Due to the use of kits with multiplex amplification, it is necessary to have a lower amount of EDTA in the sample so as not to interfere with the Mg 2+ present in the reaction. If regular TE buffer is used to dilute the sample, an imbalance will be observed in the DNA profile, while Low TE will have a better balance. The Low TE buffer or TE Low EDTA buffer is composed of 10 mM Tris-HCl (pH 8.0) + 0.1 mM EDTA.

    , lithium borate buffer, a similar buffer containing lithium ions in place of Tris and TBE buffer are often used in procedures involving nucleic acids, the most common being electrophoresis.

ph7.4 TE buffer=100mM/L Tris(pH7.4)+10mM/L EDTA(pH8.0) from Molecular Cloning: A Laboratory Manual


INTRODUCTION

Cellular responses to genotoxic stress include cell cycle arrest, DNA repair, changes in transcription, and apoptosis. Signal transduction pathways that begin with activation of the phosphoinositide-3-kinase-like protein kinases, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), coordinate these DNA damage responses.

ATM and ATR share sequence homology, substrates, and functions. Loss of ATM or ATR activity causes similar cellular phenotypes, including loss of cell cycle checkpoints and increased sensitivity to DNA damage (Abraham, 2001 Shiloh, 2001). One difference between these signaling pathways is that ATM primarily responds to DNA double strand breaks, whereas ATR responds to many forms of genotoxic stress, including DNA cross-links, base modifications, and stalling of replication forks. A key question is what allows ATR to respond to a diverse array of genetic insults, whereas ATM is limited primarily to double strand breaks.

An important clue to the biochemical differences between ATM and ATR came with the identification of an accessory protein that functions exclusively with ATR (Edwards et al., 1999 Paciotti et al., 2000 Rouse and Jackson, 2000 Cortez et al., 2001 Wakayama et al., 2001). This protein, Rad26 in Schizosaccharomyces pombe, DDC2/LCD1/PIE1 in Saccharomyces cerevisiae, or ATRIP in humans, binds to ATR and is required for ATR signaling. Deletion of the yeast ATRIP genes or RNA inhibition of ATRIP expression in human cells leads to nearly identical phenotypes as observed when ATR is deleted (Uchiyama et al., 1997 Edwards et al., 1999 Paciotti et al., 2000 Rouse and Jackson, 2000 Cortez et al., 2001 Wakayama et al., 2001). Importantly, ATRIP does not associate with ATM (Cortez et al., 2001). Thus, one hypothesis to explain how ATR can be activated by different genotoxic stresses than ATM is that ATRIP provides the broadened specificity. A second clue to how ATR might be activated differently than ATM came with the observation that ATR activation often requires DNA replication (Michael et al., 2000 Lupardus et al., 2002 Tercero et al., 2003). The interaction of a replication fork with a DNA lesion may be essential for activating ATR in many circumstances. In fact, ATR has increased activity during normal DNA replication in yeast cells and Xenopus egg extracts (Edwards et al., 1999 Paciotti et al., 2000 Shechter et al., 2004), and ATR is an essential gene in cycling human and mouse cells (Brown and Baltimore, 2000 Cortez et al., 2001). Furthermore, ATR phosphorylates some components of the replication fork, including the MCM helicase complex (Cortez et al., 2004).

The question remains how can a protein kinase complex respond to such diverse genetic insults as an interstrand cross-link, stalled replication fork, or double strand break? One model proposes that ATR is activated by physically associating with sites of DNA damage or stalled replication forks. Indeed, both ATR and ATRIP relocalize to intranuclear foci in response to DNA damage and a fraction of these proteins is chromatin associated (Hekmat-Nejad et al., 2000 Cortez et al., 2001 Lupardus et al., 2002 Dart et al., 2004). Observations that ATR activation is impaired in Xenopus extracts by depleting the single-stranded DNA (ssDNA) binding protein RPA, and ATR localization to foci in human cells is impaired when RPA expression is inhibited by RNA inhibition support this model (Costanzo et al., 2003 Zou and Elledge, 2003 Dart et al., 2004). Furthermore, ATRIP was recently shown to bind to RPA-coated ssDNA directly (Zou and Elledge, 2003), and DDC2 does not localize to sites of DNA damage in yeast mutants defective in the single-stranded binding protein RFA1 (Lisby et al., 2004). Thus, ATRIP may promote specific relocalization and activation of ATR in response to a variety of DNA lesions that are processed to expose ssDNA-bound to RPA.

In this study, we have directly tested this ssDNA–RPA recruitment model for activation of ATRIP–ATR. Surprisingly, our data indicate that an interaction between ssDNA–RPA and ATRIP is neither sufficient for ATRIP accumulation at DNA lesions nor absolutely essential for ATR-dependent checkpoint signaling.


MATERIALS AND METHODS

Cell lines, cell culture and CIRP KO cells

HTC75, HeLa, U2OS and 293T cells were cultured with Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% FBS and 1% penicillin/streptomycin at 32°C or 37°C and 5% CO2. HEK293T cells used for retrovirus production and transient transfection experiments. Full-length or mutant CIRP cDNAs were cloned into the pBabe based retroviral vector for mammalian expression ( 50). CIRP was tagged with S, FLAG, SBP at C terminus. Stable HTC75 cells were infected with appropriate retroviruses before puromycin selection (1 mg/ml for >7 days). For synchronization, cells were cultured in FBS-free DMEM medium for 24 h and then grown in fresh medium containing 10% FBS for >24 h and harvested at the indicated time points.

To generate CIRP KO HTC75 cells, we targeted exon 4. The sgRNA (5′-AACCGATCCCGTGGGTACCG) was cloned into the vector described by the Church Laboratory (Addgene) ( 51). Please see Supplementary Figure S1 for details about the clone. To generate CIRP KO HeLa cells, we targeted exon 2 (sgRNA, 5′-GCCATGGCATCAGATGA) and exon 7 (sgRNA, 5′-CATCGATGTTGTATTTGCAG), aiming to delete the entire region between the two sgRNA target sequences. The sgRNAs were cloned into the Lenti gRNA Blasticidin/Puromycin/Phage-Lenti-Inducible-Cas9-neo vector described by the Zhang Laboratory (Addgene) ( 52). KO clones were isolated as described ( 51, 52) and their genomic DNA extracted for sequencing. Successful KO was also confirmed by immunoblotting.

shRNA sequences were cloned into pcl-mU6 retroviral vectors. The targeting sequences for various shRNAs and siRNAs are:

SiCIRP-1 (CIRBP/HSS101939) siRNA

SiCIRP-2 (CIRBP/HSS174416) siRNA

SiCIRP-3 (CIRBP/HSS174417) siRNA

Immunoprecipitation-mass Spectrum (IP-MS)

293T cells stably expressing SFB-tagged full-length TERT were generated for tandem IP followed by mass spectrometry (IP-MS) as previously reported ( 53). The expression of TERT-SFB was confirmed by anti-Flag immunoblotting. For affinity purification, >2 × 10 8 cells were collected and lysed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 1 μg/ml pepstatin A and aprotinin, 1 mM MgCl2 and 250 U/Ml Benzonase Nuclease (EMD Chemicals) for 25 min. Cleared lysates were incubated with 200 μl anti-Flag M2 affinity beads (A2220, Sigma) for 2 h at 4°C and eluted with 3 mg/ml biotin (Sigma) for 2 h at 4°C. The eluates were incubated with 100 μl S-protein agarose beads (Novagen) for 2 h at 4°C before SDS/PAGE and MS (Taplin Biological MS Facility, Harvard University). Proteins with <3 peptides or found in control IP samples were excluded (Supplementary Table S1).

Bimolecular fluorescence complementation (BiFC) assay, Co-immunoprecipitation (Co-IP) assay and western blotting

For BiFC, proteins respectively tagged by YFPn (residues 1–155 of Venus YFP) and YFPc (residues 156–239 of YFP) were stably co-expressed in HTC75 cells for microscopy and flow cytometry analysis as previously described ( 54). For Western blotting, cells were lysed in 1x NETN buffer (1 M Tris-HCl (pH 8.0), 1 mM EDTA, 100 mMNaCl, 0.5% NP-40, 1 mM DTT and proteinase inhibitor cocktail before SDS-PAGE and antibody probing.

The antibodies used are: rabbit polyclonal anti-CIRP (ab94999, Abcam), anti-FLAG M2 Affinity Gel (Sigma, A2220), rabbit polyclonal anti-flag (Sigma, F7425), mouse monoclonal anti-GST (Abmart, M20007M), rabbit polyclonal anti-actin (GeneTex, GTX109639) and mouse monoclonal anti-GAPDH (Abmart, M20006). GST-tagged proteins were pulled down by glutathione agarose beads (GE).

Immunofluorescence (IF) and IF-fluorescent in situ hybridization (IF-FISH)

Cells grown on glass coverslips were fixed for 15 min on ice in 1x PBS (pH7.4) containing 4% paraformaldehyde, incubated in permeabilization solution (0.5% Trition-X 100, 20 mM HEPES, 50 mM NaCl, 3 mM MgCl2 and 300 mM Sucrose) for 10 min, followed by a second permeabilization for 30 min at RT after washes in 1x PBS. The coverslips were then blocked in 3% goat serum plus 0.1% BSA in 1x PBS followed by incubation with primary antibodies (overnight at 4°C) and secondary antibodies (1 h at room temperature). Primary antibodies include rabbit polyclonal anti-CIRP (ab94999, Abcam), mouse monoclonal anti-coilin [IH10] (ab87913, Abcam), mouse monoclonal anti-HA (H9658, Sigma), mouse monoclonal anti-TRF2 (OP129, Calbiochem). Secondary antibodies include fluorescein-conjugated goat ant-rabbit/mouse IgG (DyLight549, LK-GAR5492, Liankebio) and goat anti-rabbit/mouse IgG (DyLight488, LK-GAM4881, Liankebio). For IF-FISH, an additional incubation with PNA-TelC-FITC probe (Panagene) was conducted at 37°C for 2 h. Coverslips were mounted with Vectashield Mounting Medium containing 0.5 μg/ml DAPI and examined on a Nikon Ti fluorescence microscope.

Telomere repeat amplification protocol (TRAP) and IP-TRAP

Cells were cultured at indicated temperature (32°C/37°C, respectively) and harvested at various time points for straight TRAP assay or immunoprecipitation followed by TRAP (IP-TRAP) as previously described ( 17). The products were resolved on polyacrylamide gels (8%) and visualized with Gel Red (Biotium). Relative telomerase activity was calculated using the ImageQuant software (GE Healthcare).

Protein purification and electrophoretic mobility shift assay (EMSA)

Bacterially expressed GST-tagged CIRP and DKC1 were purified with glutathione-conjugated agarose beads, and eluted in 50 mM Tris (pH 8.0) buffer containing 50 mM reduced glutathione (GE). For EMSA, purified CIRP-GST proteins were incubated with 32 P labeled T7 capped TERC probe (Ambion) (please see Supplementary Materials for details regarding probe preparation). Binding reactions were performed at 25°C for 30 min in binding buffer (100 mM NaCl, 10 mMTris (pH7.5), 5% (w/v) glycerol, 1 mM MgCl2, 1 U of RNasin Ribonuclease Inhibitor (Promega) and 0.3 pmol of 32 P-labeled TERC probe). The products were resolved on 5% (wt/vol) native PAGE gel (100 V, 0.5x TBE, 180 min), visualized on a Typhoon PhosphorImager (General Electric Company) and quantified using ImageQuant (Amersham Biosciences).

Telomeric restriction fragment (TRF) analysis

Average length of telomeres was determined using the TRF assay analysis as described previously ( 12). Isolated genomic DNA was digested with Hinf I and Rsa I and resolved on 7% agarose gels (2 V/cm, 27 h). The denatured and dried gel was hybridized with 32 P-labeled oligonucleotides [(TTAGGG)4], exposed to a PhosphorImager screen ( 55) and quantitated on a Typhoon PhosphorImager (General Electric Company).


Spending time and money just optimizing assays and not generating usable data is super-frustrating, especially when you have limited resources. Monolith has ways to help you start with the target labeling step, and with optimizing your assays conditions so you can get to actionable results sooner.

Your success starts with choosing the right labeling strategy
The online and interactive Protein Labeling Assistant gives you the best labeling recommendations for your target protein and your instrument optics.

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MO.Control 2 software makes it super easy to check the effects of buffers on your interactions. Take advantage of the ability to run 24 capillaries to screen up to 6 different buffer conditions in duplicates and with no-ligand controls in the same run. Read the technical note for an example of how easy it is to find the right buffer conditions.

Find the optimal buffer conditions in less time
Use the Buffer Exploration Kit, a 96-well plate loaded with ready-to-use buffers commonly used in MST assays so you don’t have to pre-mix them yourself. It makes buffer screening less time-consuming.

Get instant quality checks
Key quality parameters such as aggregation, photobleaching, and low signal-to-noise are constantly monitored for you. If additional optimization is necessary, you’ll get recommendations on how to correct these issues.



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