The cell plate was created from one ml of the culture, transferred to a micro tube and centrifuged at 10,000×g for 10 min at 4 °C. One ml of PBS buffer was used to wash the cell plates. Cells were then resuspended in 100μl of 0.5M EDTA, pH 8.0 and boiled in hot water for 10 min at 100 °C. The sample was then centrifuged at 10,000×g for 10 min at 4 °C. The clear supernatant was transferred to a new micro tube. Boiling cells with 0.5M EDTA is the best method known to date for the isolation of crude PIA from staphylococcal cell surface [11 ]. The crude PIA quantification was performed by a colorimetric method as described elsewhere [12 ]. Briefly, 50 μl of the crude PIA was transferred to a micro tube and mixed with 25μl of 80% w/v Phenol solution (Sigma-Aldrich) and 1 ml of concentrated sulphuric acid was added. The solution was kept at room temperature for 10 min, and absorbance was read at 490nm. Normalization of the amount of PIA was performed by dividing by the number of cells used for extraction.
Phenol solution
Manufactured by Merck Group
Sourced in Germany, United States
Phenol solution is a laboratory reagent that serves as a solvent and chemical intermediate. It is a clear, colorless to light pink liquid with a characteristic odor. Phenol solution is commonly used in various analytical and synthetic procedures in scientific research and chemical industries.
Automatically generated - may contain errors
Lab products found in correlation
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Sulfuric acid, by Merck Group (4 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Sudan 3, by Merck Group (2 mentions)
Benedict s reagent, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Glucose, by Merck Group (2 mentions)
Sodium carbonate, by Merck Group (2 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Digital sonifier, by Emerson (1 mentions)
Tween 80, by Merck Group (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cytosine β d arabinofuranoside ara c, by Merck Group (1 mentions)
Nb300 141, by Novus Biologicals (1 mentions)
Basal eagle s medium, by Thermo Fisher Scientific (1 mentions)
Wnt8a, by R&D Systems (1 mentions)
27 protocols using phenol solution
1
Isolation and Quantification of PIA
2
Photometric Lactose Quantification
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Lactose content was determined using the photometric method (16 ). A mass of 0.5 g of sample was dissolved in distilled water and the volume was made up to 100 mL in a volumetric flask. A volume of 0.4 mL of the sample was pipetted into a test tube and 0.4 mL phenol solution (Sigma-Aldrich, Merck) was then added with proper mixing. A volume of 2 mL of concentrated sulfuric acid (Thermo Fisher Scientific) was then added and mixed immediately using vortex mixer (Spinix vortex shaker 3020; Tarsons Products Pvt. Ltd) for 30 s. After 10 min, when the temperature reached 30 °C, the absorbance of the solution was measured at 490 nm using the blank solution as reference. Reagents were added to the blank solution containing 0.4 mL of distilled water instead of the sample. Calibration curve was constructed by plotting the absorbance against the concentration of lactose in µg/mL and the mass fraction of lactose in the sample was calculated from the following formula:
where γlactose is the concentration (µg/mL) of anhydrous lactose in the analysed solution as read from the calibration curve and 0.5 is the amount of the sample taken.
where γlactose is the concentration (µg/mL) of anhydrous lactose in the analysed solution as read from the calibration curve and 0.5 is the amount of the sample taken.
3
Isolation and Analysis of tRNA Associated with Recombinant Protein
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The fraction of the purified MBP–SelU protein (purity ~99%) was dissolved in the 400 µL of storage buffer (20 mM Tris-HCl, pH 7.4, 25 mM NaCl) and mixed with an equal volume of phenol solution, equilibrated with 10 mM Tris-HCl, pH 7.4 (Sigma-Aldrich, Poznan, Poland), vortexed and centrifuged at maximum speed for 15 min. Then, the clear aqueous phase was transferred into a new eppendorf tube, treated with 400 µL of a phenol–chloroform mixture (1:1), and centrifuged as above. The aqueous phase was transferred into the new tube and washed twice with chloroform (400 µL) to remove the phenol residues. The nucleic acid present in the aqueous phase was precipitated with 2.5 volumes of 99% ethanol with the addition of 0.1 volume of 3 M sodium acetate, pH 5.0. The obtained pellet was washed with 70% ethanol and air dried. The same procedure was repeated for the isolation of tRNA from the pure MBP protein to check whether the MBP tag bound the nucleic acids (control isolation). The amount of isolated tRNA was assessed spectrophotometrically after measuring the Abs260.
For the analysis of the full-length tRNAs associated with the MBP–SelU, the protein sample (200 µg) in the storage buffer was thermally denatured (5 min at 95 °C) and centrifuged at maximum speed for 15 min at 4 °C. The supernatant was collected and analyzed using UPLC-PDA-ESI(-)-MS.
For the analysis of the full-length tRNAs associated with the MBP–SelU, the protein sample (200 µg) in the storage buffer was thermally denatured (5 min at 95 °C) and centrifuged at maximum speed for 15 min at 4 °C. The supernatant was collected and analyzed using UPLC-PDA-ESI(-)-MS.
4
Bacterial LPS Extraction Protocol
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B. theta LPS preparations were performed by Biswa P. Choudhury at the UCSD GlycoAnalytics Core. A cell pellet from 4 L confluent culture of each B. theta strain was suspended in Milli-Q water and mixed with an equal volume of 90% phenol solution (Sigma, 328111). The suspension was stirred continuously and maintained at 68°C ± 2°C for 30 min. After cooling in an ice bath, suspensions were centrifuged at 3500 rpm at 10°C for 45 min and the upper layer removed to a clean Falcon tube. The remaining layers were extracted again with an equal volume of water for 30 min, cooled, and centrifuged as before. The upper layers were pooled and dialyzed (1000 MWCO, regenerated cellulose tubing) against 4 L of water for 4 days, replacing the water twice per day. The dialysate was lyophilized, resuspended in water, and subjected to ultracentrifugation at 105,000 × g for 4 hr. The pellet was resuspended in water, treated with DNase I, RNase A, and proteinase K, followed by another round of ultracentrifugation as above. The resulting pellet was resuspended in water and lyophilized.
5
Bacterial Membrane Lysis and Biomolecule Extraction
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After induction, centrifuge 1 ml bacteria culture solution in a 1.5 ml centrifuge tube and remove the suspension. Re-suspend the pellet in 100 µL Buffer L, containing 10 mM Tris-HCl (pH 7.4) and 10 mM Mg(OAc)2. Then destroy the bacterial membrane by adding 100 µL phenol solution (Sigma).38 (link) Pipet out the aqueous layer and directly deposit it into native PAGE gel or scan under AFM. To prepare samples for denaturing PAGE gel or gel purification, 10 µL NaOAc (3 M, pH 5.2) and 200 µL ethanol were added to 100 µL aqueous layer, followed by an ethanol participation in dry ice to get rid of salts. Alternatively, the cell pellet was re-suspended in 15 ml Buffer L and sit in an ice-water bath. Cells were lysed by sonication (without phenol) with Branson Digital Sonifier (10% amplitude). Sonicating for 5 s and stop 5 s; and repeat over until total of 10 min. 1 ml lysates were centrifuged at 16,000×g for 30 min to remove the cell debris and the upper layer was diluted 40 times with TAE/Mg2+ buffer for AFM imaging.
6
Quantifying Biofilm Exopolysaccharides in Sub-MIC TC Biofilms
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Biofilm exopolysaccharides in sub-MICs TC-treated biofilms were quantified using the standard phenol sulfuric acid method [46 (link)]. Biofilms were allowed to develop in the presence or absence of sub-MICs of TC in a 96-well polystyrene plate at 37 °C for 24 h. After incubation, the planktonic supernatant was carefully removed, and the biofilms were washed with sterile PBS. Deionized water (20 µL), 5% phenol solution (20 µL) (Sigma Aldrich, St. Louis, MO, USA), and 98% sulfuric acid (100 µL) were added to the wells and incubated at 90°C for 30 min [47 (link)]. The absorbance was measured at 492 nm using a microplate reader, and the concentration of biofilm exopolysaccharides was determined using a standard curve generated using different glucose concentrations (0, 50, 200, 500, and 1000 µg/mL).
7
RNA Extraction from Log Phase Cells
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First, 10 ODs of log phase cells were harvested and resuspended in phenol solution (P4682; Sigma-Aldrich), SDS, and buffer AE (10 mM Tris·Cl and 0.5 mM EDTA, pH 9.0). Samples were incubated at 65 °C for 30 min and the phase-separated supernatant was washed with chloroform and precipitated in isopropanol overnight. The precipitate was washed with 70% ethanol and dissolved in water. RNA samples were then purified with the RNeasy Mini Kit (Qiagen).
8
Flaxseed Oil Cake Characterization
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Flaxseed oil cake (FOC) was kindly donated by ACS Sp. z o.o. (Bydgoszcz, Poland) and cold-pressed flaxseed oil was obtained via a cold press technique (Olejarnia Niwki, Zwoleń, Poland). According to the manufacturer’s information, the proximate composition of FOC was as follows: 80.5% solids content, 42% protein content, 28% carbohydrates content, 6.3% fiber content, 6.1% fat content, 4.5% ash content. Sodium dodecyl sulphate (SDS), ethanol (96%), hydrochloric acid, Tween 80, Sudan III, sodium hydroxide, Benedict’s reagent, phenol solution (5%), sulfuric acid (96%), glucose, bovine serum albumin were purchased from Sigma Aldrich (Darmstadt, Germany). All reagents were of analytical grade.
9
Neuronal Cell Culture and Analysis
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Powder of Minimum Essential Medium (MEM), fetal bovine serum (FBS), horse serum (HS), penicillin-streptomycin (PS), B-27™ supplement, L-glutamine (L-Gln), Antibiotic-Antimycotic (AA), TRIzol reagent, Lipofectamine 2000, Alexa Fluor 488, 555 IgG secondary antibodies, and 4′, 6′-Diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). Neurobasal® medium, Eagle’s basal medium (BME), and N-2 Supplement were purchased from Gibco (Grand Island, NY, USA). Poly-l -lysine (PLL), glutamate, Cytosine-β-d -arabinofuranoside (AraC), bovine serum albumin (BSA), sucrose, formaldehyde solution, chloroform, phenol solution, and IWR-1 were from Sigma-Aldrich (Saint Louis, MO, USA). WNT3A recombinant protein was purchased from PeproTech (Rocky Hill, NJ, USA). WNT8A and WNT9B were from R&D Systems (Minneapolis, MN, USA). Power SYBR Green Master Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-Tau (sc-5587) and anti-MAP2 (sc-56561) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-TUJ1 antibodies (802001 and 801202) were purchased from BioLegend (San Diego, CA, USA). Anti-GFAP (GTX108711), anti-NeuN (GTX30773) antibodies, and rabbit IgG (GTX26702) were from GeneTex (Irvine, CA, USA) or Novus (NB300-141, Centennial, CO, Canada) for cryosection staining.
10
Comprehensive Immunoassay Reagent Protocol
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Phenol solution (90%), Quillaja Saponin (Catalog# S2149), silver nitrate, sodium carbonate, sodium bicarbonate, sodium acetate, Tween 20, DMSO, fetal bovine serum, and casein were obtained from Sigma Aldrich. Tamarind seed polymer (Xyloglucan, Glyloid 2A) was purchased from Sumitomo Pharma, Japan. Laemmli Sample Buffer (2×) and Precision Plus Protein Dual Color Standard were purchased from Bio-Rad. Trichloroacetic acid, 99%, Bovine Serum Albumin Standard Pre-Diluted Set, Coomassie R-250, and phosphate-buffered saline were purchased from ThermoFisher. TMB ELISA Substrate and TMB stop (Abcam, Cambridge, UK), cross-adsorbed goat anti-mouse IgA, IgG1, and IgG2a, HRP-conjugated (Invitrogen, Waltham, MA, USA), ExpressPlus PAGE Gel, 4–20% (Genscript, Piscataway, NJ, USA), Thiazolyl blue tetrazolium bromide (MTT, Trenton, NJ, USA), 98% (Acros Organics, Geel, Belgium) and Brucella Selective Supplement (Oxoid) were used in this study.
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