Cell-Based ELISA tests now enable precise measurement of the phosphorylation status or expression profiles of target proteins (biomarkers) in whole cells. In this post, a review of high performing cell-based assays is made.
Cell Signalling: Ras-Raf-MEK-ERK pathway research tools
The Ras-Raf-MEK-ERK Pathway is an important cell signalling route with many implications for cancer biology and therapeutic development. Dis-regulation of some of this pathway’s proteins expression and phosphorylation status are observed in about one-third of all human cancers. Access to specific tools to study this pathway is essential to a better understanding of its role in cancer for novel drug development. If you are working on this topic, you’ll be interested in taking a look at the range of reagents on offer for a wide variety of applications.
Cell Signalling: MAP Kinase Signalling with phospho ELISA test
The mitogen-activated protein kinase (MAPK) signaling pathway is activated by a number of extra and intracellular stimuli including cytokines, growth factors, and hormones as well as stressors such as oxidative and ER stress. This pathways plays a key role in the regulation of many cellular processes including proliferation, differentiation, the stress response, motility, growth, differentiation, survival, and death. Abnormal MAPK signaling may contribute to increased or uncontrolled cell proliferation and/or resistance to apoptosis. To study this complex pathway, several tools are available, from the pathway specific arrays for an initial screen, to phospho-specific ELISA tests for individual target validation. This post aims at helping you to easily identify tools to explore this pathway in your samples (from arrays to phospho-ELISAs). However, I could not start without showing you once more one of these eye-catching illustrations of cell signalling pathways. I’ll let you explore it to dig out the MAPK protein cascade among all of them (a kind of Where’s Wally for the researcher !).
MAP Kinase cell signaling pathway – but which ELISA test or array should you choose to study it?
Post-Translational Modifications (PTM) of Tubulin
In this post, I’d like to take a look at the current understanding of tubulin PTMs, that include tyrosination/detyrosination, Δ2-tubulin formation, acetylation, phosphorylation, ubiquitination, glutamylation, and glycylation. This is inspired by contribution provided by Cytoskeleton Inc., who are experts in this domain.
Protein post-translational modification analysis
Protein post-translational modifications (PTMs) are part of a complex regulatory network that controls physiological and pathological cellular processes. In this post, new user-friendly assays are introduced to help Life Scientists gain a deeper understanding of these mechanisms involved in protein biology.
58 different human RTKs in one phosphorylation array
The Receptor Tyrosine Kinases (RTKs) are an important family of cell-surface receptors transmitting extracellular stimuli (ex. NGF, PDGF, FGF, EGF, Insulin…) into intracellular protein-tyrosine kinase activity, which subsequently induces a signal-transduction cascade. This signal transduction pathway leads to the regulation of cellular proliferation, differentiation, and metabolism, but also to gene expression modulation and Ras activation (a GTPase switch protein).
Monoclonal antibodies validated for PTM 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.
Ready-to-Use ELISAs to study Transcription Factors DNA binding
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. [Read more…]
Biomarkers and shear stress
Costaining of pAkt-1S473 (red) and PECAM-1 (green) after 30 min flow-adaptation and 5 min ortho- (D) or retrograde flow (E). Image was obtained using Cat. No. 039200-301-J34L for p-Akt staining.
Research nowadays aims at working on models as similar as possible to the real physiological status. This includes the modification of cell culture conditions, For example, one should perform cell culture under “real” oxygen levels (e.g. hypoxia, normoxia, physioxia). For circulating cells, shear stress is a key factor, as cells behave in a different way depending on whether they are cultured under static or dynamic conditions. [Read more…]
Post-translational modifications regulate Ral GTPases
RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).
In their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.
You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to get in contact through the form below.
Kits to measure RalA activation
- G-LISA RalA Activation Assay Biochem Kit (colorimetric format)
- RalA Activation Assay Biochem Kit (bead pull-down format)
If you’d like to get an overview about what’s available in the small G protein field, take a look at this Small GTPase product guide.