Luciferase is the general term given to a class of oxidative enzymes that catalyze reactions that give off light, a process known as bio-luminescence (Fig. 1). In biology, researchers can take advantage of this reaction and use it as a readout for various biological processes. This has perhaps been exploited most in luciferase reporter cell lines where a promoter region from a gene of interest is placed immediately upstream of the coding sequence for luciferase. In this system, transcriptional activation of the gene of interest leads to a level of luciferase expression that is proportional to the level of gene activation.
Today, I’d like to give you an overview about methods in actin research with validated R&D products and kits which will allow you to measure binding to actin and effects on the polymerisation dynamics of actin.
Actin functions as one of the major cytoskeleton structures. It is 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. [Read more…]
The term Autophagy was introduced by Christian de Duve during the Ciba Foundation Symposium on Lysosomes – which was held in London in February 1963. In 1974 he was honoured with the Nobel price in Physiology or Medicine for his pioneering research about peroxisomes and lysosomes. In 2016, once more, a pioneer in the field of autophagy research won the Nobel price: Yoshinori Ohsumi, a Japanese researcher, whose findings “led to a new paradigm in our understanding of how the cell recycles its content”.
Autophagy (Autophagocytosis) describes the fundamental catabolic mechanism during which cells degrade dysfunctional and unnecessary cellular components. This process is driven by the action of lysosomes and promotes survival during starvation periods as the cellular energy level can thus be maintained. [Read more…]
Immunotherapy represents a field in Drug Discovery which is quickly developing and leading to significant progress in treatments of a number of diseases, especially cancer. The approach is based on inducing, enhancing, or surpressing an immune response. Therapeutic manipulation of immunopathways has led to promising clinical results . The first therapeutic antibodies directed against the checkpoint receptor PD-1 have been already brought to the market (Nivolumab, Pembrolizumab) by Bristol Myers Squibb and Merck/MSD respectively, and approved for the treatment of diverse cancer types.
Today, I would like to review tools to build up a comprehensive assay set up for cell based inhibitor screening on PD-1 / PD-L1/PD-L2 binding. [Read more…]
ADCC is a simple but important mechanism for the immune system to target diseased or infected cells. Antibodies bind to specific antigens on the surface of the target cell (see Fig 1). PBMCs or natural killer (NK) cells, express Fc receptors on their cell surface and act as the effector cells. Interaction between the Fc region of the antibody and the Fc receptor induces the effector cell to degranulate, releasing IFN-γ, granzymes, and other cytotoxic compounds that lyse the target cell.
ADCC is not only a natural part of the adaptive immune response, but animal experiments have shown that it can also be seen as an important mechanism of action of therapeutic monoclonal antibodies (1), including the breast cancer drug trastuzumab, and rituximab, a drug used to treat diseases which show overactive, dysfunctional, or excessive numbers of B cells (e.g. lymphomas).
Cell lines to build up cellular ADCC screening assays
To enable researchers to build up a cellular ADCC screening system, BPS Biosciences have developed 2 reporter cell lines, which can replace NK cells or PBMCs in such a cellular assay (see Fig 2). The system is based on Jurkat cells that stably express human FcγRIIIa (CD16a), the receptor for the Fc region of human IgG. The FcγRIIIa on the Jurkat cells binds to the IgG on the surface of the target cell. This crosslinking causes the Jurkat cells to activate NFAT transcription, which induces the expression of luciferase and can be easily detected using the ONE-Step™ Luciferase Detection Reagents.
The effectiveness of ADCC depends on how well the effector cells are activated after the engagement of FcγRIIIa. Human FcγRIIIa displays dimorphism at amino acid 158 – one allele (V158) encodes a high Fc affinity receptor variant, while the other (F158) encodes a lower Fc affinity receptor variant. BPS offers 2 different ADCC cell lines expressing either of these Fc receptors to allow selective antibody binding analyses using each type of receptor.
- ADCC Bioassay Effector Cell, F variant (Low Affinity)
- ADCC Bioassay Effector Cell V variant (High Affinity)
Get more information about our ADCC cell lines – just leave your questions or comments in the form below!
(1) Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV; Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets; Nat Med. 6 (4): 443-446 (2000)
Subscribe to thematic newsletters on your favourite research topics.
Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through Mass Spectrometry or immunoblot with linkage specific antibodies, which are cumbersome. LifeSensors has developed the UbiTest assay, a more definitive method for demonstrating the ubiquitylation linkage of a protein, which is to couple immunoprecipitation of polyubiquitylated protein with digestion by a linkage specific deubiquitylase prior to immunoblot analysis. An increased signal for the unmodified substrate or a decreased signal of polyubiquitylated substrate at high molecular weight after K48/K63 specific DUB treatment is a clear indication that the protein was K48/K63 ubiquitylated. [Read more…]
Are you working on autophagy,? Or on GPCR de-orphaning? On the MAP kinase pathway or cellular metabolism? Maybe on pathways related to cellular metabolism?
Several compounds are known as modulators of these pathways and research fields and can be used as useful tools to study and characterize specific steps. Nonetheless, it is quite time consuming and expensive to order all these chemicals from different sources. Targeted compound libraries represent a convenient alternative. [Read more…]
Small GTP-binding proteins such as RhoA, Rac1, and Cdc42 are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. They bind and hydrolyze GTP, thus being switched from the activated form to the inactivated form (Fig 1).
The most prominent family of small G proteins is represented by the Ras superfamily of proteins. The Rho subfamily belonging to this superfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia. [Read more…]
Extracellular adenosine 5′-triphosphate (ATP) is released by dying and damaged cells, and it acts on many immune cells to promote inflammation. On the other hand, the unphosphorylated
metabolite, adenosine, functions as an anti-inflammatory molecule. Two extracellular ecto-5´-Nucleotidases, CD39, and CD73, convert extracellular ATP to ADP/AMP and AMP to adenosine, respectively, leading to elevated levels of extracellular adenosine (Fig 1). [Read more…]
The Silicon Rhodamine-like (SiR) technology has significantly contributed to the recent development of DNA and cytoskeletal analysis by live cell imaging.
In 2014, two new Silicon Rhodamine-like (SiR) fluorescent probes were released for studying actin & tubulin by live cell imaging. SiR-Actin and SiR-Tubulin are fluorescent probes compatible with most microscopes (including super-resolution settings) that directly stain actin & tubulin without the need to transfect cells with vectors expressing fluorescently labeled Actin or Tubulin. The two original dyes were successfully followed by a new SiR-DNA probe in order to visualize DNA in living cells.
The existing SiR stains have a λabs of 652 nm and a λem of 674 nm to be used with the Cy5 filter (Fig 1).
However, the continuously growing number of researchers using these stains asked us whether stains with different biophysical properties would be made available. In other words, they were asking “is there another colour to allow for double staining e.g. of Actin and Tubulin in living cells?”