Plasmin activity assays were performed as previously described with minor modifications18 (link). E. faecalis were grown overnight in tryptone yeast media. OD600 was normalized by dilution to 0.1, and samples were diluted 10x in the final reaction. Bacteria were incubated with 250nM human glu-plasminogen (Haematologic technologies) with or without 10mM TXA (Fisher) for two hours at 37°C. A final concentration of 4nM uPA or pro-uPA (Biovision) were added and incubation proceeded for 20 minutes. A final concentration of 6μM plasmin-specific fluorogenic substrate (H-D-Val-Leu-Lys-AFC, AnaSpec) was added immediately prior to a 30-minute kinetic fluorescent read at 380/500nm. For assays of uPA activity, PLG was omitted and a substrate specific to uPA (Z-Gly-Gly-Arg-AMC, Bachem) was utilized. When assays were performed in human plasma, fluorescence was read for 120 minutes total. Plasmin or uPA activity is expressed as initial reaction velocity calculated from change in fluorescence over time during the initial phase of the reaction, when pseudo-first order kinetics determine the rate.
Pro upa
Manufactured by Abcam
Sourced in United States
Pro-uPA is a recombinant human pro-urokinase protein. Urokinase-type plasminogen activator (uPA) is an enzyme that plays a role in the degradation of the extracellular matrix, which is involved in processes like tissue remodeling and cell migration. The pro-uPA product is the zymogen, or inactive precursor, form of uPA.
Automatically generated - may contain errors
2 protocols using pro upa
1
Plasmin Activity Quantification in E. faecalis
2
Quantifying Aptamer-Protein Interactions by MST
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Microscale thermophoresis (MST) experiments were performed on a Monolith NT.115 (NanoTemper Technologies, Munich, Germany) using standard capillaries. Measurements were performed at an MST power of 60% and excitation power ranging from 20 to 60%. In our work, 5′-cyanine 5-labeled aptamers were diluted to 20 nM in BPsT, refolded as previously described and used as the fluorescently labeled binding partner at a constant concentration. Serial dilutions of HMW- and LMW-uPA ranging from 0.1 to 333 nM were prepared in BPs-T. For pro-uPA (BioVision Inc., Milpitas, USA), mouse uPA (Abcam plc, England) and tissue-type plasminogen activator (tPA, EMD Millipore Corp., Burlington, VT, USA) concentrations ranged from 0.1 to 500 nM. In the research, 10 µL of refolded aptamer and protein dilutions was mixed and incubated for 30 min. Samples were measured by the Monolith NT.115. The recorded fluorescence was normalized to the Fnorm per mill and fitted utilizing the KD formula derived from the law of mass action by MO Affinity Analysis software (NanoTemper Technologies GmbH, Munich, Germany) version 2.3 [33 (link),34 (link)]. All measurements were performed as triplicates.
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