Monoclonal validated for Post-Translational Modification studies

Knowing the Human genome better has allowed major advancements in Personalised Medicine. Nowadays, we can know (if we want) the likelihood to develop a given disease and/or how we will react to different pharmacological treatments. Examples of this include diseases like breast cancer (for diagnosis or estimation of likelihood) and lung cancer (for response to treatment), to name just a few.

That said, our genotype does not have the last word. Research in the last couple of decades has shown the power of other regulatory mechanisms, that may enhance or diminish the effect that our genotype will have on our health. Starting from basic healthy life styles, to other more subtle mechanisms, our genotype defines us, but not completely. Above genetics, we have epigenetics… and everything at the protein level. This post will focus on Post-Translational Modifications (PTM), because, after all, it’s the proteins that are the final effectors of a given response to a treatment or to an environmental stimulus.

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How to get clear best-quality data in IP and WB protocols?

Immuno-precipitation & Western Blots often suffer from heavy  /light chain blotting, contamination, and ongoing interferences. This can prevent from obtaining biologically-relevant data in a given experimental model, especially when the target of interest has a size similar to the IgG molecules that may remain. Getting rid of these “artificial” bands can be tricky. In this post, you will discover the robustness of the TrueBlot® products when facing this issues.

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Oxygen and miRNAs in Cosmetology and Dermatology

A recent review by Nadim et al. casts some light on a Cosmetology and Dermatology, where circulating biomarkers, though studied to a certain extent so far, are yet unknown for many skin models. (1) A first element to have in mind when considering the skin-related experimental model is the “Oxygen level”. Oxygen levels may contribute to different findings, and the in vitro models used so far may not be so physiologically relevant as initially thought.

Hypoxia and miRNAs

Tissue oxygenation (which is a major part of the cell microenvironment) regulates the expression of the microRNAs called oxymiRs. OxymiRs may be categorized into three groups:

  1. microRNAs whose expression is directly modied by the Oxygen partial pressure,
  2. microRNAs whose expression is indirectly (pH, metabolites, etc.) modified by the Oxygen partial pressure, andpO2 percentage levels in various tissues
  3. microRNAs that target mediators of Oxygen sensing pathways to regulate biological networks for cell survival.

Examples of miRNAs important in skin physiology include miR-17, miR-21, miR-24, miR-27, miR-29b, miR-99 family, miR-125, miR-146a, miR-155, miR-203 and miR-205, among many other.

For example, in injured tissue, disruption of the vascular supply is associated with a low oxygen partial pressure, or hypoxia, which induces the expression of specic microRNAs referred to as HypoxamiRs (included in group (1) of the oxymiRs classification).

miR-210, known as the master HypoxamiR, is robustly induced under hypoxic conditions in nearly all kinds of cells. Under hypoxia, miR-21 also induces angiogenesis by targeting PTEN, leading to activation of AKT and ERK1/2 signaling pathways.

Transfection Factors and microRNAs

Other transcription factors such as p53 and NF-kB have been shown to affect the expression of microRNAs under hypoxia/anoxia conditions.

Numerous studies have demonstrated the modulation of microRNA expression and particularly that of the miR-200 family with oxidative stress due to excessive ROS levels.

Up to now, very few studies have shown the role of the intermediate Oxygen level (physioxia) in regulating microRNA expression in skin cells. Taken together, these results strongly support the idea that physioxia should be an important criterion in determining the microRNA expression level and consequently protein expression and skin functions.

Therefore, when trying to understand the role of microRNAs in skin models, it is important to choose a cell culture system that is as physiological as possible, and have the tools to analyse the different biomarkers (no only microRNA, but also signaling pathways, secretome, etc).

Should you like to have more information on this review, or know what we can do for you to support you with cell culture technologies with controlled oxygen levels or to support you for your biomarker discovery studies, do not hesitate to contact me by leaving a message below.

Source:

(1) Nadim M. et al. “Physioxia and MicroRNAs As Key Factors in the Skin Microenvironment” (2015) FSCC Magazine, Vol. 18 – #1, pp: 35-43

Array profiling to study PPAR-alpha in progenitor cell

Peroxisome proliferator-activated receptor alpha (PPAR-alpha also known as NR1C1)  regulates a myriad of biological processes. It is a key modulator of lipid metabolism.

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services)

Raybiotech C-Series Arrays by tebu-bio (ready-to-use kits and lab services).

Vergori, L. et al. have shown in murine models how PPAR-alpha regulates endothelial progenitor cell maturation and myeloid lineage differentiation via a NADPH oxidase-dependent mechanism. (1) All the data described in this publication suggest that PPAR-alpha, in murine models, is a critical regulator of recruitment, homing and maturation of Bone Marrow-derived progenitor cells.

This conclusion was made (in part) possible with the analysis of secretome markers by using C-Series profiling arrays for the analysis of bone marrow-derived cells in PPAR-alpha wild-type vs. KO mice.

RayBio® Membrane-Based Antibody Arrays (C-Series) are tools for screening and comparing expression levels of many cytokines between samples. C-series Arrays are available as ready-to-use kits or lab services. To ensure top quality in the data obtained, Raybiotech’s service providers successfully complete training and certification programs to receive the “RayBiotech Certified Array Service Providers” yellow award.

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012.

tebu-bio’s services also cover Quansys BioSciences and FullMoon BioSystems technologies for outsourcing protein profiling and quantification.

Interested in studying the secretome biomarkers relevant in your model for Cardiovascular diseases?

Leave your comment or contact us!tebu-bio: European RaybioTech's Certified Laboratory service provider

Source:
(1) Vergori L. et al. “PPARa regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice” (2015) Stem Cells – 33 (4) :1292-303. DOI: 10.1002/stem.1924.

Phosphorylation studies made with Antibody Arrays

Oropharyngeal squamous cell carcinomas (OSCCs) can be either Human papillomavirus (HPV)-positive or HPV-negative. Profiles of druggable Receptor Tyrosine Kinases (RTKs) are different in both groups, as shown in a paper by Cortelazzi, B. et al. The authors chose a cohort of 17 HPV-positive and 59 HVP-negative Formalin-Fixed OSCCs, in order to study E5 expression and RTK alterations. RTK activation was explored in further 12 Frozen OSCCs.

HPV-positive and HPV-negative OSCCs showed different RTK profiles, including differences in E5 and HER2 levels, as well as in HER3 activation and heterodimerisation (HER3/EGFR, also seen for HER2/EGFR). PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases.

The authors stated that, for HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. They also indicate that in HPV-negative cases, HER3 may be a promising druggable biomarker that would deserve further investigation. Finally, PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCCs, particularly in HPV-positive/PI3KCA-mutated OSCCs.

This study was possible in part thanks to an approach based on arrays to detect multiple biomarkers in biological samples at the same time, followed by validation using simplex technologies. These experimental approaches are known for cytokine profiling but also exist for phosphorylation studies (RTKs, EGFR, mTOR phospho-pathways…).

To ensure top quality in the data obtained, you might outsource your biomarker profiling and validation to certified service providers fully trained. tebu-bio: European RaybioTech's Certified Laboratory service provider

In Europe, tebu-bio laboratories (France) were among the first laboratories in the world to be certified by Raybiotech in 2012 but also by Quansys BioSciences and FullMoon BioSystems technologies. The Biomarkers team will be proud to support you to publish novel discoveries by providing innovative discovery & validation tools.

Normalisation assays in biomarker studies – all’s well that starts well

The normalisation of quantitative assay data is critical when interpreting effective biological system status. With cells grown in culture and lysed, a simple total protein determination such as the Bradford assay (developed by Marion Bradford at the University of Georgia in 1976) can be enough by giving an estimate of the total cellular proteins. However, this type of measurement, along with Lowry and other dye binding assays, can be prone to errirs due to various factors such as detergent, chelators… Standardization also involves the use of a protein of interest. Here again, the protein used is crucial for the accuracy of the overall assay.

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Mambalgin 1 – Blocking ASIC channels in pain pathways

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels which are activated by extracellular protons. They belong to the ENaC/Deg superfamily of ion channels. Up to now 6 members of the ASIC family have been identified: ASIC1 – ASIC 4, and the splice variants ASIC1a, 1b and 2a, 2b. The ASIC family members are trimeric and can be made up of different combinations of subunits. All ASICs are expressed in the peripheral nervous system. ASIC1a, 2a, 2b and 4 are expressed in the central nervous system. ASICs are promising drug targets for treating a wide variety of conditions linked to both the CNS and PNS especially implied in pain pathways.

Structure of Mambalgin-1

Schwarze Mamba

Fig. 1: Dendroaspis polylepis polylepis

Mambalgin-1 (from Smartox, now available through tebu-bio) was initially isolated by Sylvie Diochot and collaborators from the venom of the black mamba (Dendroaspis polylepis polylepis, Fig. 1).

Mambalgin-1 belongs to the family of three-finger toxins (Fig. 2) and has no sequence/structural homology with either PcTx1 or APETx2. Mambalgin-1 differs from mambalgin-2 by one amino acid. Both have demonstrated a similar activity.

Mambalgin 1 – a blocker of ASIC1 channels implied in pain pathways

It has been shown that Mambalgin-1 is a potent and selective blocker of ASICs implied in pain pathways (1, 2). Mambalgin-1 rapidly and reversibly inhibits recombinant homomeric ASIC1a (IC50=55 nM) and heteromeric ASIC1a+ASIC2a (IC50=246 nM) or ASIC1a+ASIC2b channels (IC50=61 nM) but also human channels hASIC1b (IC50=192 nM) and   hASIC1a+hASIC1b (IC50=72nM).

Mambalgin-1 has no effect on ASIC2a, ASIC3, ASIC1a+ASIC3 and ASIC1b+ASIC3 channels, as well as on TRPV1, P2X2, 5-HT3A, Nav1.8, Cav3.2 and Kv1.2 channels. Thus Mambalgin-1 can be used as a selective inhibitor for the above mentioned homomeric and heteromeric ASICs.

Interested in further information? Please contact me through the form below.

If you’d like an overview of the complete venomous toxins available, take a look to our special page: Synthetic peptide toxins ideal for studying ion channels

References:

(1) S, Dichot et al., Black mamba venom peptides target acid-sensing ion channels to abolish pain, Nature. 490 (7421), pages 552-5 (2012)

(2) Wen M., et al., Site-specific fluorescence spectrum detection and characterization of hASIC1a channels upon toxin mambalgin-1 binding in live mammalian cells. Chem Commun. 51 (38), pages 8153-6 (2015)

Keen to work with the best quality nucleosomes?

Histone Structure- tebu-bio

Fig. 1: Structure of a mono-nucleosome.

Nucleosomes are basis units of DNA packaging in eukaryotic cells. A core particle (mono-nucleosome) consists of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are made up of 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. H1 histone, the so-called linker histone, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.). [Read more…]

8 criteria for selecting your ELISA kits

Biomarkers specialists are often asked to select an ELISA kit for researchers: with thousands of ELISA references available on the market, the choice can be tricky regarding proteins for which several kits available.

When researchers have to choose a new ELISA kit, the price is regularly the first parameter of selection. But my experience with long term projects shows that it should in fact be the very last one…

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Focus on Actin detection and Actin binding proteins

Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.

In the coming weeks I’d like to give you an overview of methods in actin research with validated R&D products and kits (actin polymerisation, and G-F actin ratio detection in cells); I also invite you to take a look at a post recently released about actin visualization: Focus on Actin staining and visualization.

In today’s post, let’s concentrate on a method which allows measuring actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity. [Read more…]