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…]

Click Chemistry: What it means for biologists

A few months ago I read a very nice blog post from our friends at TriLink Biotechnologies giving the chemist’s perspective on the excitement surrounding “Click Chemistry” and how it can be used to make non-natural, yet functional DNA and RNA. Some of the terms in that post such as 1,3-dipolar cycloaddition are oriented more towards chemists. Here’s a more biologist-friendly explanation of Click Chemistry: [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.

Discovery of new biomarkers… 3 tips regarding controls

One of the recurrent questions that we get at the Biomarkers team at tebu-bio is on what controls should be included in a given experiment. Either if the experiment is done by researchers in their lab, or if we collect their samples and perform the analysis in our lab, a good design starts by using the most convenient controls.

One of the controls is related to the study itself. In this sense, definition of what a control population is, and how we want to study it vs. a cohort of patients has been discussed elsewhere in a proteomics post. Today, we will put our spotlight on the “technical” controls, i.e. those related to the technique itself.

Control # 1 – positive control

Obvious. We need to check that the technology we are using is able to detect what we want to detect. And before starting with unknown samples, we need to check that it works in samples we know well.Multicoloured wells - Blog Thumbnail

Ideally, a positive control should be as similar as the samples we want to analyse. In this sense, samples for a given health state (be it with a disease or not), are commercially available, or they can be found if not yet available. tebu-bio has a network of collaborations with private companies that can provide validated samples, fulfilling all ethical and clinical criteria.

Untitled

Background can be an issue…only if you are not able to detect it. If you can see it, then you can either modify your protocol, or discard that sample. Picture shows an example of what can be seen with slightly-hemolysed plasma.

Alternatively, we can use recombinant or chemically synthesised controls. If we take the example of recombinant proteins (either in pure form or spiked in biological samples), there are many which are commercially available, or they can be made upon demand. Here it is important that the recombinant protein is very similar to the one found in an organism, including glycosylations and other post-translational changes. In this sense, for many control proteins, HEK293 is preferred over E.coli as an expression system.

Control #2 – negative control

Water. Or PBS. Or not?

Ideally, a negative sample should be as similar as to our case samples as possible. Meaning that it has the same clinical and biological parameters than our samples of interest…

Commercially available samples mentioned previously can be a good approach. For immunological studies involving cell culture supernatants, it is important to include a control with the culture medium only, as FBS can affect the specificity of the assay and can render false-negatives due to background.

Background due to FBS is not detected by technologies such as ELISAs or bead-based, whereas it is detected in optical-based technologies such as arrays and Q-plex.

Control #3 – technical replicates

Every technology has an inherent coefficient of variation (CV).

Genomic technologies usually are under 5 % CV. Immunoassays are around 10 to 25 % (or even more). This means that, for some biomarkers where the difference between healthy vs. disease is small, CV may hide the relevance of these biomarkers. This is especially dramatic in studies related to signal transduction, where differences are usually very small.

6-plicates in an antibody array.

6-plicates in an antibody array.

A way to make sure about whether a result comes from real biology or artificial CV is the performance of replicates.

Triplicates (or even 4-plicates) have been popular with ELISA users. Nowadays, however, most researchers perform duplicates, and repeat the analysis of the sample if the results are very discordant. This approach is quite practical, and still allows to have accurate results in a sensible way (i.e. not doing 4-plicates for every sample!).

Antibody arrays in the market usually have replicates spotted onto the same slide (from duplicates to 8-plicates), which can be considered as semi-independent technical replicates. Therefore, there is no need, in most cases, to perform additional technical replicates.

In any case, every project is different, so we are continously advising our customers on what is the best approach for one given study. From its design to the technology best suited to get the best results, we are glad to contribute to the advance of the understanding of biomarkers in several diseases.

Wondering what controls to include in your experiment? Don’t hesitate to contact us!

Focus on Actin staining and visualization

Actin serves as one of the major cytoskeleton structures. It is a crucial component involved in a plethora of processes in cell biology:  stabilizing the cell shape, cell movements (e.g. cell migration)  and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group.

A brief reminder: G-actin polymerizes to form F-actin

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Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)

Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1).  Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).

In the coming weeks I will give you an overview about methods in actin research with validated R&D products and kits (actin binding and actin binding proteins, actin polymerisation, and G-F actin ratio detection in cells).

 

Today I will focus on methods to visualize actin in fixed or living cells – which belong to basic experimental set-ups in Cell Biology.

Actin staining of fixed cells

Actistain photo

Fig. 2: Swiss 3T3 fibroblasts stained with ActiStain 488 (green), Dapi (blue) and Anti Vinuclin (orange). Provided by Cytoskeleton Inc.

Often fluorescent phalloidins are used to stain actin in fixed cells. Phalloidin belongs to the group of phallotoxins produced by the mushroom Amanita phalloides (death cap mushroom). The natural toxicity of Phalloidin is due to its stabilizing effect on F actin in cells. Based on its affinity for F-actin and coupled to a fluorescent dye, it can be used to visualize F-actin.

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability.

Results of staining of Swiss 3T3 cells with ActiStain 488 are shown in Fig. 2.

Live-cell imaging of Actin

Live-cell imaging of actin has been quite tricky as far as actin labelling is concerned – either cells had to be transfected with vectors carrying the genetic information for fluorescently tagged actin or actin binding proteins or, labelled actin had to be micro-injected to single cells.

Actin

Fig. 3: 3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts. Provided by Spirochrome.

Together with Spirochrome, tebu-bio launched in Europe the first tool to directly label actin in living cells with no need to transfect or micro-inject anything.

SiR-Actin is a cell permeable compound which stains F-actin in living cells. The stain is composed of a photostable silicon rhodamine-like (SiR) dye which can be used with standard Cy5 settings and a component (Jasplakinolide) which specifically binds to F-actin (Fig. 3). SiR-actin is compatible with Super-Resolution Microscopy like Stimulated Emission Depletion [STED] and Structured Illumination Microscopy [SIM].

f you would like to get an overview about the results SiR-actin users got so far, please have a look at my recent blog: User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging.

 

 

 

Interested in our Phalloidin and/or SiR-based stains?

Leave your comment or request in the form below.

 

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!

9 pathway-specific screening assays in Immunotherapy

The immune system is a system of cells and organs whose function is to defend an organism from foreign pathogens. With the ability to mount a response against virtually any foreign material and return to a quiescent state following neutralization of the threat, this fascinating organ system displays remarkable specificity and plasticity. To achieve this, there is a multifaceted balancing act between the many activators and suppressors which maintains homeostasis of the body’s perhaps most complex organ system.

[Read more…]

PEI transfection patents to expire

July 7, 2015 marked the 20-year anniversary of the filing dates of both the U.S. patent and European patent limiting the use of PolyEthylenImine (PEI) as a transfection reagent. Coincidentally, both U.S. and European patents generally have a term of 20 years from the filing date.

Many academic researchers have been ignoring these patents and/or have been sharing protocols online and publishing articles explaining how cost-effective and simple PEI-mediated transfection can be. For example, the most commonly used PEI, catalog nr. 07923966-2 (Polysciences), is a linear form with molecular weight of 25,000 Da. The 2 gram bottle of PEI powder can be used to make a few liters of transfection reagent, so depending on which protocol is used the cost can be about 0.01% that of commercially-available transfection reagents.

PEI structure. Image source: https://en.wikipedia.org/wiki/Polyethylenimine

PEI structure. Source: Wikipedia.

PEI is a simple polymer with an amine group and two carbon spacer that is thought to bind to DNA to produce positively charged particles that can enter the cell.

Some of the negative aspects of PEI compared to more sophisticated transfection reagents are:

  1. PEI tends to be rather cytotoxic. While PolyJet™ DNA In Vitro Transfection Reagent  is composed of proprietary bio-degradable polymers designed to greatly reduce cytotoxicity, standard PEI has been shown to induce apoptosis in a variety of human cells.
  2. PEI is difficult to dissolve. Linear PEI is solid at room temperature and somewhat soluble in hot water and low pH. A typical protocol might involve dissolving 8mg of PEI in 25mL of water, a process that may take many hours or days and/or result in a non-uniform solution that cannot be sterile filtered.

To solve this problem, Polysciences has released the a much more soluble hydrochloride salt form (catalog nr 07924765-2), called Polyethylenimine “Max”, (Mw 40,000) – High Potency Linear PEI. This MW 40,000 form corresponds to the MW 25,000 polymer length in free base form, with the salt accounting for the molecular weight difference.

PEI max structure.

PEI max structure.

Looking through the literature one might reach the conclusion that PEI is a universal transfection reagent suitable for transfecting cultured cells or use in vivo.

Here’s a summary of some of the earlier scientific articles using PEI for various transfection applications:

Nucleic Acid Cell Type Publication
68-mers Hepatocytes ref
antisense oligos Neurons ref
BAC DNA mouse (in vivo) ref
DNA 293E ref
DNA Adult neural stem cells ref
DNA Brain derived cells ref
DNA chicken (in vivo) ref
DNA CHO cells ref
DNA Cos-1 ref
DNA Cos-7 (nuclei) ref
DNA Embryonic neurons (in vivo) ref
DNA Fetal mouse liver ref
DNA HeLa cells ref
DNA Human monocytes ref
DNA HUVEC ref
DNA L929 cells ref
DNA LNCaP cells ref
DNA Mouse brain ref
DNA Mouse lung ref
DNA Mouse lung ref
DNA Murine adult neural stem cells ref
DNA Ovarian carcinoma cells ref
DNA Postmitotic neurons ref
DNA Pseudocystic tumor cells ref
DNA Rat (in vivo) ref
DNA Rat brain ref
DNA Rat fetal hypothalamic cells ref
DNA Rat kidney ref
DNA Rat kidney ref
DNA WERI-Rb1 retinoblastoma ref
DNA Xenopus tadpole brain ref
dsRNA and siRNA Snail, Biomphalaria glabrata ref
modified siRNA Murine melanoma cells ref
siRNA in vivo ref
siRNA Pancreatic cancer cells ref
YAC HT1080 cells ref

 

 

 

 

 

 

 

 

 

In addition to use as a direct transfection reagent, PEI is used for a variety of “combination” gene delivery methods. Adenofection for example makes use of PEI to permit delivery of large plasmids/BAC/YAC into cells by non-covalently coupling of the DNA to an adenovirus. A variety of other approaches make use of modified (e.g. PEGylated) PEI for gene delivery or have targeted PEI to specific tissues with antibodies or proteins.

In the list of applications, mRNA delivery into cells seems conspicuously absent. Indeed at least one paper indicates that PEI-mediated transfection of mRNA is very inefficient. For mRNA transfection, researchers have better success with the Stemfect™ RNA Transfection Kit or the mRNA-In Transfection Reagent.

Transfection reagents by tebu-bio

HA tagged beta-tubulin cDNA was delivered into CHO cells. HA-beta-tubulin (Green) – Endogenous alpha-tubulin (Red).

 

Leave your comment below to share your experience with PEI!

 

 

 

 

 

 

 

 

 

 

 

 

 

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…]