After 48 h of incubation, the bacterial culture broths were centrifuged at 8000 g for 20 min at 4°C. The cell‐free supernatant was 20‐fold concentrated at 70°C for 24 h and centrifuged at 100,000 g for 1 h at 4°C to remove the precipitate. The resulting supernatant was then filtered through a 0.2 μm pore‐size membrane (Millipore). The filtrate was fractionated through stepwise ultrafiltration using 10 and 1 kDa cutoff discs (Millipore) to yield high molecular weight (HMW, >10 kDa), middle molecular weight (MMW, 1–10 kDa), and low molecular weight (LMW, <1 kDa) fractions. The fractions were lyophilized and stored at −70°C until use. Each fraction (100 mg) was dissolved in 20 mM Tris–HCl (pH 7.0) and loaded onto a Superdex 30 prep‐grade (pg) column (200 ml) pre‐equilibrated with the same buffer in a Biologic Duo‐Flow FPLC system (Bio‐Rad) at a rate of 1.5 ml/min. Eluents were monitored by measuring the absorbance at 210 nm, and each peak fraction was pooled, lyophilized, and stored at −20°C until use. Based on the standard curve obtained using proteins/peptides of known sizes (Figure S1 ), 1–10 kDa peptide fractions were pooled and used for further analysis.
0.2 μm pore size membrane
Manufactured by Merck Group
Sourced in Switzerland, United States
The 0.2 μm pore-size membrane is a laboratory filtration product. It is designed to filter particles and microorganisms from liquids with a high degree of precision, retaining materials with a minimum size of 0.2 micrometers.
Automatically generated - may contain errors
Lab products found in correlation
Biologic duoflow fplc system, by Bio-Rad (1 mentions)
Phosphate buffered saline solution, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Amicon centrifugation tube, by Merck Group (1 mentions)
5 protocols using 0.2 μm pore size membrane
1
Peptide Fractionation and Purification
2
Algal Extract HPLC-MS Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Accurate weights (1 mg/mL) from each dried algal extract were separately prepared HPLC-grade methanol, filtered through a 0.2 μm pore size membrane (Millipore) and degassed by sonication before injection. Full loop injection volume (10 μL) of each sample was applied onto the chromatographic column. Detailed preparation of standard solutions for UPLC–MS quantification can be found under Supplementary Material .
3
Proteomics Data Analysis Pipeline
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the reagents were of analytical purity grade. Dithiothreitol (DTT), iodoacetamide (IAA), and LC-MS grade acetonitrile were purchased from Fluka (Buchs, Switzerland); urea, trifluoroacetic acid (TFA), and formic acid from Merck (Zug, Switzerland); TRIS and acetone from Sigma (Buchs, Switzerland); and sequencing-grade endoproteinase LysC and porcine trypsin from Promega (Dübendorf, Switzerland). Phosphate-buffered saline solution was from Gibco (Life Technologies, Zug, Switzerland) and sterile filtered through 0.2 μm pore size membrane (Millipore, Zug, Switzerland).
Normalization, imputation, statistical tests, and Spearman rank correlations were calculated using base R with following additional packages: vsn, MSnbase, and limma. All protein expression data are provided in theSupplementary File proteinGroups_DE_test.xlsx . Lasso feature selection was performed using the R package glmnet (version 4.0-2). Community clustering was calculated by the GLay app [26 (link)] from Cytoscape [28 (link)]. Graphics art were designed in Photoshop using figures that were produced with R, Excel, and Cytoscape.
Normalization, imputation, statistical tests, and Spearman rank correlations were calculated using base R with following additional packages: vsn, MSnbase, and limma. All protein expression data are provided in the
4
Purification of Pythium insidiosum Biomass
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Pythium insidiosum Pi-S strain was subcultured on Sabouraud dextrose (SD) agar for 1 week. Several small pieces of agar-attaching hyphae were transferred to a 250-ml flask containing 100 ml of SD broth for shaking incubation (∼100 rpm) at 37°C for 7 days. The hyphal material was removed by filtration through a 0.2-μm-pore-size membrane (Merck Millipore, USA). The harvested hyphae were extracted to obtain SABH, as previously described (24 (link), 54 (link)). The cell-free culture broth was concentrated using an Amicon centrifugation tube (10,000 molecular weight cutoff [MWCO]; Merck Millipore) to obtain CFA, according to the reported protocol (24 (link), 54 (link)). Both SABH and CFA were stored at –30°C until use.
5
Isolation and Characterization of Legionella pneumophila Secreted Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L. pneumophila strains were grown to an OD660 of 1.8 to 2.0 in a shaking incubator at 230 rpm at 37°C. Supernatants were isolated by centrifugation of the cultures at 5,000 × g at 4°C, followed by filtration through a 0.2-μm-pore-size membrane (EMD Millipore). Supernatant protein was concentrated 25× (vol/vol) as follows. Isopropanol (100 ml) was added to 50 ml of culture supernatant and incubated at −20°C overnight. Precipitated protein was centrifuged at 10,000 × g for 30 min at 4°C, and the resulting pellet was washed twice in 70% ethanol and resuspended in 2 ml of PBS with added cOmplete protease inhibitor cocktail. Sample volumes were normalized to the measured bacterial optical density, diluted in SDS-loading buffer, and analyzed by immunoblotting as previously described (21 (link)). Primary antisera were used at the following concentrations: CelA, 1:5,000; ChiA, 1:10,000; and ProA, 1:5,000 (in 1% milk [wt/vol]–Tris-buffered saline [TBS-T]). The secondary antibody, goat anti-rabbit horseradish peroxidase antibody (Cell Signaling Technology, Inc.), was diluted 1:10,000 in 1% milk–TBS-T.
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!