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.

[Read more…]

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.

[Read more…]

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

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…

[Read more…]

Basics about insulin and dedicated research tools

Diabetes is a major health concern. And its research can be a nightmare sometimes. tebu-bio strive to offer a comprehensive range of research tools and services to study Obesity, Diabetes, and Metabolic syndrome (including pancreatic islet cells), and tools to unravel signaling mechanisms in insulin secretion. Anyhow, it might be good, though, to go back to the basics from time to time. Let’s remember our graduate courses (more or less years ago) about Insulin and its biological roles.

[Read more…]

New way for derivation / maintenance of naïve human PSCs

Newly added to the Stemolecule portfolio are three small molecules which support a new approach for the derivation and maintenance of naïve human pluripotent stem cells. These three newly identified small molecules, WH-4-023, SB590885 and IM-12, are all kinase inhibitors.

small-molecules-03

ReproCELL is currently the only stem cell reagent company able to supply all components of the 5i/L/A (5 inhibitors/human LIF/Activin A) media supplement formulation needed for the derivation and maintenance of a “naïve” or ground-state of pluripotency of human cells as referenced in the October 2014 Cell Stem Cell paper by Theunissen et al. out of Dr. Rudolf Jaenisch’s Lab at MIT’s Whitehead Institute. The paper is titled “Systematic identification of culture conditions for induction and maintenance of naïve human pluripotency.”

Stemgent Stemolecule WH-4-023 (SRC Inhibitor)IM-12 Structure

Stemgent Stemolecule SB590885 (BRAF Inhibitor)

Stemgent Stemolecule IM-12 (GSK-3β Inhibitor)

 

The addition of these three new small molecules consolidates a 5i/L/A media supplement offering in one place. 5i/L/A media supplement consists of 5 kinase inhibitors; WH-4-023 SRC inhibitor, SB590885 BRAF inhibitor, IM-12 GSK-3β inhibitor, PD0325901 MEK inhibitor, Y27632 ROCK inhibitor, recombinant human leukemia inhibitory factor (LIF) and Activin A. The 5i/L/A media supplement allows for the derivation of and conversion of human PSCs to a naïve state of pluripotency hypothesized to be the human equivalent to the mouse ground state of pluripotency.

The Theunissen paper demonstrates the conversion of traditional human epiblastic pluripotent stem cells to a ground state of pluripotency thought to be equivalent to the traditional mouse embryonic stem cell state. The human equivalent to the mouse ground state of pluripotency is termed naïve. Naïve human PSCs differ from traditional human epiblastic ESCs in that human ESCs are FGF/BMP signaling dependent (equivalent to mouse epiblastic stem cells). While naïve PSCs are LIF/Stat3 signaling dependent (equivalent to traditional mouse ESCs). Both traditional mouse ESCs and naïve human PSCs demonstrate higher single cell viability when passaging. Likewise both cell types demonstrate enhanced proliferation when compared to epiblastic stem cells. These attributes make naïve human pluripotent stem cells a good choice for genetic manipulation and gene targeting applications.

Related products used in the paper that researchers may be interested in include: Human recombinant FGF-basic growth factor, CHIR99021 and doxycycline.

Keep cool… corticosterone and stress

Corticosterone is a glucocorticoid secreted by the cortex of the adrenal gland in response to stimulation by adrenocorticotropic hormone. Corticosterone is a major indicator of stress in non-human mammals. Glucocorticoids, such as corticosterone, guide fundamental processes associated with converting sugar, fat, and protein stores to useable energy; inhibiting swelling and inflammation, and suppressing immune responses following a stress event.

Measuring Corticosterone

corticosterone

Comparison of the EIA kit described in this post with traditional RIA, using 1 ul mouse tail bleed samples.

Competitive immunoassays, such as RIA and EIA methods, are the typical means for measuring levels of corticosterone in biological matrices. Most RIAs or EIAs require solvent extraction techniques to measure serum or plasma corticosterone levels, however extraction may be very difficult or impossible with mouse samples due to the large volumes of plasma or serum required for most extraction protocols. [Read more…]

Ready-to-Use ELISAs to study Transcription Factors DNA binding

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA

TFACT™ DNA-BINDING ELISA KITS » OCT2 TFACT™ DNA-BINDING ELISA by Assay Biotechnology. Source: tebu-bio.

Gene expression is regulated by different mechanisms. One of them is the binding of Transcription Factors (TF) to DNA sequences.

Traditionally, the study of TF-DNA interactions is made by several time-consuming and cumbersome: Electrophoretic Mobility Shift Assays (EMSA), Chromatin Immunoprecipitation, Western blotting, and expression of fused target and reporter genes.

ELISA-based formats now allow to have a more precise TF-DNA interaction study in addition to an ease of use.

These new tools significantly reduce the necessary runtime (within one day) and eliminate the need for harmful radioactive labeling. High sensitivity and signal-to-noise ratio are also guaranteed.

The TFact™ product-line belongs to these new ELISA-based Transcription Factor DNA binding assays. These indirect ELISAs allow an easy the detection and qualitative determination of the effects of phosphorylation on transcription factor activation profiles in a variety of nuclear and cell lysates from human, mouse and rat.

TFACT™ DNA-BINDING ELISA KITS » AML1 TFACT™ DNA-BINDING ELISA: AML1 (Phospho-Ser435)

The TFact™ AML1 DNA-Binding ELISA detects active AML1 in Hela Nuclear Extract. The Hela cells were grown 3 days in DMEM with 10% FBS and harvested for nuclear extract. The Hela cells were stimulated by PMA (200nM) before harvest.

TFACT™ DNA-BINDING ELISA Kits are available for various Transcription Factors and well-defined phosphorylated sequences: AML1, Jun, Androgen & Estogen Receptors, ATF2, CREB, FOXO, NFkB, STAT, p53, myb, SMAD, Sox …

Looking for simple but robust methods for studying TFs and the effect of phosphorylation in their activity?

Contact me for any further assistance!

More is not always better – Tech tips for ELISAs

Following our previous post on how to improve results obtained in ELISA, let’s focus today on one specific point, which is reducing background.

ELISA has many advantages, but one of the drawbacks is that, since we cannot “see” how the reaction works (in contrast to other optical-based technologies such as antibody arrays or Q-plex), high final Abs values may come from a specific signal… or be due to background.

Usually, incorporating sufficient controls in the ELISA plate will allow users to discriminate real positives from false positives (e.g. if you are using cell culture supernatant with FBS % over 1 %, it might be wise to include a medium-only control). FBS contains cytokines that can cross-react with antibodies, even if targeted to different species, in about 10 % of the cases (based on our experience at the Biomarkers team at tebu-bio).

Anyway, if you suspect that you are obtaining a high background in your ELISA, and would like to improve it for future experiments, be sure to follow these guidelines (thanks to Daniel at Raybiotech, Inc. for helpful tips & tricks!). [Read more…]

DNA repair antibodies validated by MD Anderson

A series of publications (1, 2) in the past months has raised the never-ending debate on commercial antibodies and their validity in a given experiment. At tebu-bio, having sold antibodies for more than 40 years, we are well aware that not all antibodies fit all applications, and always strive to find the best antibody for a given experiment.

In collaboration with the MD Anderson Cancer Center, Rockland Inc. has developed and validated a highly specific toolkit to analyze PARP1 in a panel of control and siRNA knockdown cell lysates by multiple immunoassays. [Read more…]