Tools, Methods & Assays

Atat1 - A new target for the development of novel analgesics

Neuropathic pain is a complex, chronic pain state that can have various causes, such as damage of the somatosensory nervous system. The treatment of this chronic pain syndrome remains challenging. Since most painkillers are ineffective on neuropathic pain, current methods mostly comprise helping the patient cope through psychological or occupational therapy.

Here we present a new target for the development of novel analgesics that could address neuropathic pain and open up a new chapter in the treatment of this condition.

The sense of touch depends upon the transformation of mechanical energy into electrical signals by sensory neurons in the skin. This conversion is thought to be mediated by a complex of proteins in which ion channels such as Piezo2 function as mechanotransducers. EMBL scientists showed that mice with a neuron specific deletion of the α-tubulin acetyltransferase (Atat1) were insensitive to mechanical pain. The results indicate that compounds disrupting Atat1 function could be developed into painkillers that will be effective for the indication of neuropathic pain and outperform existing treatment methods.

Download PDF

LiMA, a liposome microarray to systematically study protein lipid interactions

The interaction of proteins with lipids is of fundamental importance in most cellular process. To date it is studied by either testing protein-lipid binding affinity towards a very limited number of ~10-20 individual lipids (mostly phospholipids) spotted on a membrane. This is not physiological, as the lipids do not form membrane bilayers, and does not take into account cooperative mechanisms, as lipids can be probed only one-by-one and not in complex combination. Methods based on the use of artificial surrogate membranes only partially resolve these issues, and their fabrication, storage and handling are difficult, they are not readily scalable, often require non-physiological buffers with lipid-specific adjustments, use large amounts of lipids and purified proteins, which precludes their use in large and systematic analyses. Considering that a eukaryotic cell produces more than 1000 different lipid species, each with distinct properties and often acting in combination, a tool is needed that enables the study of protein-lipid interaction in a manner that is on a par with the functional genomics resources now available, i.e. a simple device to comprehensively study protein-lipid interaction in a physiological, sensitive, reproducible, cooperative and high-throughput manner. LiMA is a mircoarray chip to measure protein recruitment to membranes that satisfies this need.

Download PDF

microRNA miR-142 as pluripotent stem cell marker and differentiation regulator

Pluripotent stem cells (PSCs) play an important role especially in the areas of regenerative medicine and drug discovery. One of the challenges in working with pluripotent stem cells is to control the states of pluripotency and differentiation uniformly within one culture. The microRNA miR-142 was shown to be a switch for the differentiation state of PSCs.

Download PDF

SerpinA1 as target and companion diagnostic for Parkinson’s disease dementia (PDD)

Up to 80% of Parkinson’s disease (PD) patients can develop Parkinson’s Disease Dementia (PDD). Currently treatment options are limited and diagnosis is only possible after significant pathophysiology has established. Serpins are a broadly distributed family of protease inhibitors. Structural changes in serpins due to aberrant modifications are a likely mechanistic link to PDD pathology.

The key scientists to this invention have shown in two studies (one with 93 individuals from 3 centers & one with 102 individuals from 2 centers) that differences in the sialylation of serpinA1 isoforms can distinguish between PD and PDD patient groups and they developed an assay for the fast, reliable and high-throughput applicable analysis of serpinA1 in cerebrospinal fluid (CSF) patient samples.

The research highlights the importance of serpinA1 in PDD as it might provide a novel target for developing treatments, act as a companion diagnostic for presymptomatic PDD treatment and could be established as the first predictive diagnostic marker for PDD.

Download PDF

SP3 - ultrasensitive, rapid, unbiased and efficient protein purification for proteomics

As downstream applications such as mass spectrometry have advanced, proteomics have reached an important impact in both research and development and clinical applications. However, the precedent step, the sample preparation, still represents a bottleneck in the workflow in respect of duration and loss of sample material. SP3 allows for ultrasensitive (< 1 µg), rapid (< 15 min handling time), unbiased and flexible sample preparation. The whole sample preparation process can be carried out in one single pot so the loss of sample during purification is prevented. The technology can be easily automated and is therefore ideally suitable for high-throughput approaches.

Download PDF

High-throughput method for producing monoclonal antibodies

Production of new antibodies against novel targets remains restricted by high tissue culture load and low-throughput screening methods, which result in costly and inefficient processes. The technology developed at the EMBL circumvents two major obstacles to increase the mAb production throughput level, the number of tissue culture operations necessary for performing multiple fusions simultaneously using only one antigen per animal as well as screening the many thousands of culture supernatants generated by large-scale production.

Download PDF

Ultrafast, highly sensitive, fluorescent protein stain

Separation and analysis of proteins is daily routine in molecular biology laboratories. SDS-PAGE is one of the most important and most widely used methods whereby proteins are separated according to their size by gel electrophoresis. To date, however, in order to reveal the results of the separation, gels have to be washed, fixed and stained after the gel run. These procedures do not allow experimenters to follow the separation in real-time, they are very time-consuming and require several handling steps. Thus, there is a clear need for a reagent that overcomes these limitations. For the first time our novel, ultrafast and highly sensitive fluorescent protein stain offers a superior combination of sensitivity, extreme speed and unprecedented ease-of-use.

Download PDF

ESPRIT, a systematic approach for generating soluble protein variants

Proteins often express insolubly which severely limits their usefulness in areas such as structural analysis by crystallography and NMR. Several systems have been described that aim to identify soluble protein variants generated by random mutagenesis or truncation. These methods usually involve fusion of a C-terminal “solubility reporter” (e.g. GFP, CAT or beta galactosidase). The tag used in the methods described above are large and thus enhance the solubility profile of the fusion product. The solubility of the thus created fusion protein is highly dependent on the solubility phenotype of the tag used. By using a smaller tag the solubility influence of the tag is reduced leading to a reduction of false positives.

Download PDF

Cell-based method for the analysis of protein-protein interactions

Protein-protein interactions are crucial for virtually all cellular processes. Therefore analysis of such interactions is becoming increasingly important in molecular biology, biochemistry and computational biology. Furthermore, disturbed protein-protein interactions can contribute to diseases rendering the identification of protein-protein interaction inhibitors highly desirable in drug discovery. Traditional technologies used for analysis of protein-protein interactions share the major drawback that the experimental set-up is highly artificial, questioning the physiological relevance of such interactions in vivo. The technology presented here allows for reliable analysis of intracellular protein-protein interactions based on a translocation principle.

Download PDF

Novel coating for efficient cellular uptake and retention of small gold nanoparticles (GNPS) for radiosensitisation

Radiotherapy in combination with surgery and chemotherapy is a fundamental part of cancer treatment with about 50% of cancer patients receiving radiotherapy during the course of their disease. In radiotherapy tumor control correlates with the dose given to the tumor. The sensitivity of tumor cells to radiation can be increased by introducing particles of high atomic number into tumor cells. Gold nanoparticles (GNPs) are particularly versatile, however, a major obstacle is that with current methods their optimum size for cellular uptake is limited to 50nm. A higher rate of radiosensitisation, and thus improved dose modifying factors, are expected from smaller GNPs, and there is a clear need for a method that allows efficient internalisation of small GNPs in tumor cells to improve patient outcomes. Our novel DNA-coating allows for efficient cellular uptake of particles as small as 5-10nm.

Download PDF

Novel biomarkers for the detection of testicular carcinoma in situ and derived cancers in human samples

Virtually all testicular cancers originate from the same precursor, the carcinoma in situ (CIS) cell. If left untreated, CIS will invariably progress into testicular cancer. Unfortunately, the disease is rarely diagnosed at this asymptomatic stage, since it hitherto has required a testicular biopsy to identify CIS. The aim of the current work is to develop and validate a non-invasive diagnostic test for early detection of testicular cancer based on identification of pre-invasive CIS cells in semen samples.

Download PDF

Rapid detection of multi-resistance in bacteria by HPLC/MS

Early and targeted antimicrobial therapy improves patient outcome, decreases cost and prevents the spread of microbial resistance which is becoming a world-wide health problem. Fast, actionable results are often critical – e.g. for sepsis patients mortality rates increase by 7% for each hour without adequate treatment. Our approach directly tests for the presence extended spectrum ß-lactamase (ESBL) enzymatic activity, the enzyme responsible for multi-resistance in bacteria. It uses high-performance liquid chromatography (HPLC) and mass spectrometry (MS) and is based on our newly discovered principle of inverse temperature dependence of ESBL mediated antibiotic hydrolysis. The assay provides actionable results within only 90 minutes, the fastest such assay available to date. It thus provides a significant increase in sensitivity with simultaneous substantial decrease of in-laboratory turnaround time. Bacterial growth is not required for the assay thus also patients pre-treated with antibiotics can be analysed and treatment success can be monitored.

Download PDF

Phasing macromolecular structures by UV-induced damage

This invention relates to a novel method to phase macromolecular crystal structures using damage induced by UV radiation (UV-RIP). Compared to existing techniques this method shows the advantage that it can be performed on a single crystal of the native protein and introduces only specific changes. The technology can be used independently of a synchrotron.

Download PDF

Close