Optimal substrate design for protease screening!

EnSens assays, a new protease activity assay technology, which are produced by  Enzium, were recently introduced to the European market. A number of  assays are available from catalog, such as activity assays for MMP-2, MMP-9, MMP-14, MMP-25, ADAM10, ADAM17, Factor Xa, Furin, and Thrombin.

But what about if what you’re looking for is not available? In this post, we’ll take a look at how you can get an optimal substrate for your protease of interest, even if it is not yet obtainable from catalog.

EnSens technology

First, let’s take a look at the EnSens assay technology itself. It’s based on a non-FRET technology and makes use of a protein substrate expressed in E. coli which is a modular dumbbell comprising two domains separated by a linker that contains a highly specific protease recognition sequence. One domain is the dye-binding domain, and the other is a blocking domain. When the linker is cleaved by a protease, separating the two domains, the binding domain is free to bind dye, constraining it and causing it to fluoresce (see Figure below).

EnSens principle

EnSens assays overcome limitations of FRET based protease assays

  • the substrate can incorporate full recognition site sequences – in FRET assays the recognition sites usually have to be shorter which can result in a number of false positives and negatives because the recognition site might not be highly selective.
  • The substrate is an scFv-based protein (single-chain variable fragment, an artificially generated antibody fragment) which confers high stability in vitro and in vivo.
  • The EnSens se-Red fluorophore is resistant to photobleaching up to 48 hours
  • The long emission wavelength (665nm) reduces interference from other assay components
  • The assay works stably in wide range of pH, salt, & solvent conditions
  • Substrate and fluorophore are separate in the systems, so the fluorophore is constantly renewed in solution
  • The system is characterized by high signal-to-noise ratios: 6-20x
  • The read-out can be done in standard plate or cuvette fluorimeters
  • The EnSens assay gives repeatable and reproducible kinetic activity curves from reagent to reagent

and finally (and this is where it gets interesting for non-catalog items!):

  • The substrate is modular, allowing easy swapping of protease recognition sites – thus customized substrates can be obtained if you know the recognition site of your protease of interest

So, you can get your customized protease substrate!

In other words: If you want to screen for modulators/inhibitors of a protease with a known recognition site, you simply need to provide the sequence of the site – and the substrate and the complete EnSens assay can be designed for you in cooperation with Enzium.

Interested in a highly specific protease assay for your screening? Get in touch through the form below, I’m sure a tailored solution can be found to match your needs exactly!

 

5 reasons to prefer custom rec. protein production to commercial

Many recombinant proteins (rec. proteins) are now commercially available. And many of them, mainly secreted soluble proteins, are biologically active, so there shouldn’t be any need to look for alternatives such as custom productions.

So the question is, why should you prefer a special production?

Well, there are several reasons (and they’re good ones too!). [Read more…]

Direct access to Biomarker Profiling identification tools

Profiling tools have an increasing interest for identifying new biomarkers. Different tools are available, from classical gene profiling on DNA chips, RT-qPCR arrays, Protein arrays for secretome or transcriptome, mRNA arrays and finally miRNA arrays. All these techniques sometimes require specific material for reading arrays, and some bioanalytics to extract valuable markers of interest.

Expand your knowledge – a new visionArrays - Blog Thumbnail

The increasing focus on these tools is obvious as they help to expand scientific knowledge on sometimes well-known pathways. Why restrict your analysis to classical markers instead of checking the impact on more than 100 biomarkers… at a similar cost? Value of results is obvious as well, as it offers a convenient way to identify original pathways.

The “Start smart” attitude

Facing a new project is always a stressful situation. Biomarkers chosen are generally based on literature, which may lead to duplicating, more or less, already existing information. Searching for innovative pathways looks more like a cherry-picking, highly risky strategy. At the end, this may lead to rather conservative conclusions. A striking example concerns Western blots (WB) which everybody knows may be time consuming, rather expensive and for sure limited in number for a given project. Nobody will ever make the decision to perform more than 100 WB to explore all the potential targets available. Lab’s budgets can’t survive such a strategy, and probably the time allocated to a project cannot suffer such delays…

Protein profiling on secretome or transcriptome now allows you to study more than 1000  targets from a single sample at once. Pricing is equivalent to 10 to 20 WB traditional WB. Results obtained are of top value as they immediately orientate research focus to appropriate pathways without having the risk of missing crucial information. This helps to speed up projects, focus research towards original biomarkers and at the end deeply differentiate published results.

But not everybody has the appropriate material to perform these assays, nor is used to handling these arrays. This generally needs adapted scanners to perform readouts and once results are obtained, spots need to be further analyzed to guarantee final quality. It requires some skill. One can understand the reluctance to jump into these technologies when they are only of occasional need.

So where’s the solution?

For these reasons, tebu-bio has developed over these last years a complete Profiling – Biomarker identification platform that offers researchers access to various solutions. With a complete offer including RT-qPCR arrays, protein arrays for soluble or signal transduction markers, mRNA and miRNA arrays, we help researchers to immediately identify biomarkers involved in their domains. This information, which often appears as the initial step in project management, drives studies in the appropriate direction, rapidly and in a cost effective way.

The service process is extremely simple. After defining the most appropriate solution regarding goals of interest, your samples are tested and results sent back. Time frame is generally 2 to 4 weeks depending on starting material. A dedicated project manager is always available for any questions throughout the project. There is no license involved in these studies, which are performed in our own, European-based labs near Paris.

For those who don’t have access to cell culture facilities, our cell culture platform is also ready to collaborate, with access to a large stock of primary cells.

And the next step…

Well, if you’re curious to know how this could help boost your research, get in touch to see exactly how it can work! Just leave your question or comments below.

Why not outsource your plasmid preps ?

Plasmid preps are to science what salt is to cooking. It’s so common that nobody notices, but nothing can be done without. There is no mention of this step in any published paper, nobody mentions them in any activity report, no comments are made on time spent on it.Screen Shot 2014-12-31 at 4.55.17 PM

But let’s face it: it takes time, as simple and as usual that it is. Some days spent, here and there. It’s so well planned in your weekly agenda, it’s such a habit, that you don’t even think about it. It’s just routine. When you’ve done a plasmid prep a couple of times, there’s nothing new to learn about it. It’s like riding a bike, once you know you don’t even notice any more.

But it takes time.

[Read more…]

MDR1 activity enhancement reduces senescence markers

During the last International Federation of Societies of Cosmetic Chemists (IFSCC) congress (Paris, Oct. 2014), Hajem N. et al (ALES Groupe) have shown that by enhancing MDR activity of fibroblasts grown in vitro, a positive effect on fibroblast cells was detected. In addition to directly testing MDR1 activity, the authors also used a genomic profiling approach to correlate the protective effect of their D-GlyOx complex with a reduction in senescence biomarkers on treated fibroblasts.

tebu-bio’s laboratories supported the Ales Groupe (LIERAC, PHYTO, CARON, Laboratoire DUCASTEL) by providing fibroblast cellular models and measuring the anti-ageing effect of their D-GlyOx complex by genomic profiling,

Source: Hajem N. et al “MultiDrug Resistance (MDR) proteins: active protective system, source of cell longevity” – International Federation of Societies of Cosmetic Chemists (IFSCC) congress – Oct. 27-30, 2014 – Paris) Poster # P-080.

6 Tips for a successful ELISA

ELISA are immunoassays widely used in biomarker detection and validation. They have been used in research and clinical settings for more than 40 years, and they allow to quantify, in a simple way, thousands of biomarkers. New ELISAs are being developed every day, either for new biomarkers or new species. They can also be used to detect contaminants in food, water or industrial processes.Raybiotech ELISA Kits by tebu-bio

Technology-wise, there are several types of ELISAs, depending on whether they are based on double (or sandwich) antibody detection, direct detection, competitive detection, etc. It is not the scope of this post to enter into details about the different formats that an ELISA can have, but if you think that you need this info, feel free to suggest that I do a post on that!

Nevertheless, the aim of this post is to give some hints on how to reach the best result for your ELISA tests, based on some troubleshooting experience with researchers and clinicians all over Europe. [Read more…]

Sircol for soluble and insoluble collagen assays!

Measuring soluble collagen in biological samples is a straightforward experiment which can be done with the Sircol soluble collagen assay using Sirius Red dye (Sircol dye) in a convenient manner (we explored this method in a recent post).  Nevertheless, up to now it has been a challenge to measure insoluble collagen.

[Read more…]

Adipokines in obesity and glucose homeostasis

Obesity is the abnormal increase in adipose tissue mass. This phenomenon increases the likelyhood of a number of diseases such as heart disease, high blood pressure, type 2 diabetes, obstructive sleep apnea, osteoarthritis, and some kinds of cancer.

Obesity is probably the leading preventable cause of death worldwide. People are considered to be obese if their body mass index (BMI) exceeds a value of 30. The mainstays of treatment of obesity remain dieting and physical exercise.

Nevertheless, adipose tissue (fat) cannot be considered a worthless batch of “lard”, but rather a fascinating vital tissue that in addition to being the body’s major energy reservoir, plays a central role as a secretory organ. Research in this field has already lead to exciting results related to both fat reduction, insulin resistance and to the development of robust tools to study diabetes / obesity.

Adipokines are factors secreted by adipose tissue which carry messages to other parts of the body. I would like to briefly introduce the role of adipokines in the regulation of body weight and glucose homeostasis. [Read more…]

How to select Hyaluronic Acid (HA) quantitative tests?

Hyaluronic acid (also called HA or Hyaluronan) is a glycosaminoglycan with unique characteristics. HA possesses elevated viscosity, enabling tissues (eye, skin, joint and synovial fluid…) to resist to physical and mechanical constraints (torsion, flexion…). Over time, when HA production is declining, tissues progressively loss these tensile properties, leading to wrinkles and fold, weak reepithelization and age-related troubles. [Read more…]

1 simple way to rapidly quantify Collagen: Sircol Collagen Assay

Collagen is a structural protein expressed in mammalian tissues (ex. skin, muscles, tendons, ligaments, cartilages, blood vessels…).  Together with Elastin and Keratin, Collagen fibres give connective properties to mammalial tissues ensuring their physiological form but also firmness, strength and flexibilityNeedless to say, Collagen is trendy… a simple search in Google using the term “Collagen” clearly shows the economical dimension of this protein in well-being.

[Read more…]