What will the Next-Gen Protease activity detection be?

Fluorescent technologies enable the measurement of protease activities and the screening of compounds influencing protease activities. Fluorescence Resonance Energy Transfer (FRET) but also TR-FRET, Q-FRET together with Time Resolved Fluorescence (TRF) and Fluorescence Lifetime (FLT) are popular assays, in which protease substrates occupy a central place. These assays are based on the specific recognition and cleavage of a peptide sequence (substrate) by the protease of interest. If the substrate is not optimally designed (or too short), experimental output can be unrelevant: low selectivity and specificity, high background, false negatives or positives…). In addition, the data can also be affected by the reaction buffer (pH, compound solvent…).This lack of relevant biological information is at the origin the emergence of “Next-Gen” fluorescent protease substrates.

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Flow cytometry intracellular staining

Intracellular Flow Cytometry enables the identification and analysis of signaling and functional markers within cells from samples containing heterogeneous cell populations. Many cell types can be thus identified through their intracellular Flow Cytometry patterns and key intracellular signaling pathways and their respective post-translational modifications can be conveniently analysed. Behind these unique performances, Intracellular Flow Cytometry require anyhow optimized immunoreagents for cell permeabilization, intracellular staining and fluorescent detection. [Read more…]

5 Tips for Flow Cytometry

Flow cytometry, even if somehow a “classical” technique, is still very valuable in Immunology. Uses of flow cytometry vary from cell identification & sorting based on membrane molecules, characterisation and quantification of secreted and intracellular molecules, as well as, more recently, analysis related to exosomes.

Following our series of technical tips for common experiments in Life Sciences (phospho-WB, secondaries, IF, ELISA, primary cell culture), we present today a series of tips to improve your flow cytometry results. [Read more…]

5 tips for successful flow cytometry results

Dr. Camilo Moncada  is Director of QC at Rockland Immunochemicals, a US Biotech leader in antibody developments. With his team of scientists, Dr. Moncada develops and validates innovative antibody-based research tools.

Flow cytometry (FCM) is an attractive experimental approach, especially when accurate monitoring of cell membrane and intracellular pathway signaling biomarkers is required.

In a technical note compiled by Dr. Moncada on tebu-bio’s initiative, he reviews 5 simple but critical experimental steps in order to design optimal FCM protocols and improve flow cytometry results.

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