The stock solutions (10–2 M) of the compound T126 were diluted to a decreased molarity, from 100 µM to 0.0001 µM, in 384-well transparent plates (Greiner 781801) with 1% DMSO: 99% buffer Hepes 100 mM, DMSO 4%, Brij 0.003%, Tween 20 0.004%, MgCl2 10 mM, at a pH = 7.5. Incubation occurred at 37 °C and the results were read after 2 h in a NEPHELOstar Plus (BMG LABTECH, Ortenberg, Germany). The results were adjusted to a segmented regression to obtain the maximum concentration in which the compounds were soluble.
Nephelostar plus
Manufactured by BMG Labtech
Sourced in Germany
The NEPHELOstar Plus is a multi-mode microplate reader that measures the scattered light intensity from particles suspended in a liquid sample. It is designed to perform nephelometric measurements, which quantify the turbidity or cloudiness of a solution.
Automatically generated - may contain errors
19 protocols using nephelostar plus
1
Solubility Assessment of Compound T126
2
Solubility Determination of Compounds
3
Nephelometric Solubility Determination
4
Solubility Screening of Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The stock solutions (10-2 M) of the assayed compounds were diluted to decreased molarity, from 300 μM to 0.1 μM, in 384 well transparent plate (Greiner 781,801) with 1% DMSO: 99% PBS buffer. Then, they were incubate at 37 °C and read after 2 h in a NEPHELOstar Plus (BMG LABTECH). The results were adjusted to a segmented regression to obtain the maximum concentration in which compounds are soluble. Digossin, prazosin and progesterone were used as reference compounds (equilibrium solubility = 84.0, 62.8 and 6.5 μM, respectively) [71 (link)].
5
Proteinase K-mediated Dissolution of PrP Fibrils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibrils in growth buffer were incubated with a 1:10 molar ratio of freshly dissolved Proteinase K (GoldBio) to rPrP94-178 monomer for 24 hours at room temperature without agitation67 (link). After the incubation period, any remaining insoluble material was pelleted by centrifugation at 16,160 xg for 5-7 minutes and the supernatant removed. The pellet was resuspended in water (for negative stain imaging) or 2 % SDS (for cryo-EM).
For a measure of insoluble character in response to Proteinase K treatment, dissolution experiments were monitored by nephelometry (Extended Data Fig. 2 ). A slurry of 100 μL of rPrP94-178 fibrils in growth buffer with the addition of 1:10 molar ratio of Proteinase K: rPrP94-178 monomer (10 μM ProK: 100 μM rPrP94-178 monomer), growth buffer with the same amount of Proteinase K, growth buffer only, or water were added to a sealed 96-well plate and loaded into a NEPHELOstar Plus (BMG Labtech). Readings in nephelometry units (NTUs) were recorded at 37 °C for 0.1 seconds every 10 minutes for 24 hours with a 2.5 mm beam at 12 % intensity. Before each reading, the plate was shaken at 300 rpm for 20 seconds.
For a measure of insoluble character in response to Proteinase K treatment, dissolution experiments were monitored by nephelometry (
6
Nephelometric Precipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nephelometry was performed using NEPHELOstar Plus equipment (BMG LABTECH, Ortenberg, Germany). Kaolin was used as the internal standard, and compounds were added to HBSS buffer as DMSO stock solution to a final concentration of 1% DMSO and a total volume of 200 μL. Precipitation or aggregation was considered significant when average values exceeded three times the standard deviation of the blanks. Blank values were omitted if they exceeded three times the standard deviation of the 24 blanks that were measured on each 96-well plate. All compounds were tested in triplo, and wells of suspected outliers were visually inspected before omitting any outliers. Data were processed in Prism version 8.0 (GraphPad, San Diego, CA, USA).
7
Nephelometric Screening of Azobenzene Propranolol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In transparent flat-bottom 96-well plates, the azobenzene propranolol analogs, under dark conditions or preilluminated with 360 nm light to PPScis, were placed at different concentrations in triplicate (10−4 M, 10−4.5 M, 10−5 M, 10−5.5 M, 10−6 M, 10−6.5 M, 10−7 M, 10−7.5 M and a blank) in aqueous buffer with 1% DMSO at least 1 h before the measurement. A Kaolin dispersion was used as a positive control.(Roessler and Brewer, 1967 (link)) Nephelometry measurements were performed with a NEPHELO star Plus (BMG Labtech, Germany) with the following settings: laser intensity 80%, beam focus 2.0 mm, and Orbital shaking of 10 s at 200 rpm before data acquisition. Results were analyzed using GraphPad Prism 8 software, plotting all available data points and plotting mean and SD values in a line chart compared to kaolin control. The linear fit (R2) of the kaolin control was above 0.99 in all cases.
8
Ligand Solubility Optimization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The stock solutions (10–2 M) of the selected ligands were diluted to decreased
molarity, from 300 to 0.1 μM, in a 384-well transparent plate
(Greiner 781801) with 1% DMSO:99% PBS buffer. These were incubated
at 37 °C and read after 2 h in a NEPHELOstar Plus (BMG LABTECH).
The results were adjusted to a segmented regression to obtain the
maximum concentration in which compounds are soluble.
molarity, from 300 to 0.1 μM, in a 384-well transparent plate
(Greiner 781801) with 1% DMSO:99% PBS buffer. These were incubated
at 37 °C and read after 2 h in a NEPHELOstar Plus (BMG LABTECH).
The results were adjusted to a segmented regression to obtain the
maximum concentration in which compounds are soluble.
9
PEG-8000 Titration Assay for Protein Stability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A stock solution of 40% (w/v) PEG-8000 in succinate-Arg buffer was prepared as the titrant. Stock protein solutions were prepared at 9.8 mg/mL in the same buffer. Samples were prepared in triplicate and loaded into a 96–well, clear, flat-bottomed plate (Greiner Bio-One, cat. 655801) using a liquid handling system (Freedom-EVO150, Tecan, Männedorf, Germany) as follows: (i) succinate-Arg buffer was dispensed into each well with volume of 100–180 μL (ii) 20 μL of protein solution was dispensed into each well to aa final concentration of 1 mg/mL; (iii) 0–80 μL of the PEG-8000 stock solution was titrated into each well to give a series of PEG-8000 concentrations from 0–16%; (iv) samples were mixed by slowly aspirating and dispensing the well contents five times (total sample volume in each well was 200 μL). Plates were examined for air bubbles and nephelometry measurements were made immediately using a NEPHELOstar Plus (BMG Labtech, Ortenberg, Germany).
10
Fungal Conidia Growth Monitoring
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We added 190 μL of broth culture media in sterile 96‐well microplates (Thermo Scientific) and completed each well with either 10 μL of conidia suspension at 106 conidia.mL−1 or with 10 μL of Tween 80 (0.045% v/v) for negative controls. For incubation, microplates were stored in a plastic box containing sterile distilled water to regulate humidity during incubation. Measures were obtained using a laser nephelometer (NEPHELOstarPLUS, BMG labtech) with a laser diode at 635 nm. At the beginning of each measurement, a 20‐s 500 rpm double‐orbital agitation step was performed by the nephelometer microplate reader (Savary et al., 2022 (link)). Each microplate was monitored four times a day for days 1–2, thrice a day for days 3–7, twice a day for days 8–14 and once a day for days 15–21 or until the signal reached saturation. For each experiment, a minimum of three replicas per strain and condition were performed for more robust estimates of growth parameters.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!