Six water samples collected from a natural wastewater treatment plant (irrigated fields) located in Wroclaw, Poland, as phage sources were centrifuged at 15,000 g for 15 min, and the supernatants were filtered through a 0.22-μm Millex-GP filter (Merck Millipore, Darmstadt, Germany, SLGP033RS). One milliliter of filtered water sample and 0.5 ml of a bacterial broth culture, grown overnight in TSB, were added to 10 ml of TSB and incubated at 37 °C for 18 h. The suspension was then centrifuged again, treated with chloroform, and filtered through a 0.22-μm Millex-GP filter (Merck KGaA, Darmstadt, Germany). This procedure was repeated three times to eliminate any bactericidal activity by contaminating chemicals. Bacteriophage presence and titer in the filtrate were assessed by the plaques test using the double-agar layer technique (Adams 1959 ). Phages were propagated from a single plaque. The phage lysate was then subjected to PEG 8000 (Acros Organics, Geel, Belgium) precipitation. The 28 environmental bacteriophages lytic on PA strains (27 named KT and one phage PA5oct) were deposited in the phage collection of the Institute of Genetics and Microbiology, University of Wroclaw, Poland.
Millex gp filter
Manufactured by Merck Group
Sourced in United States, Ireland, Germany, United Kingdom, Australia
The Millex-GP filter is a disposable sterile syringe filter designed for the filtration of aqueous solutions. It features a 0.22 μm hydrophilic PVDF (polyvinylidene fluoride) membrane that effectively removes particulates and microorganisms from the sample. The filter has a low hold-up volume and fast flow rates, making it suitable for a variety of laboratory applications.
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Lab products found in correlation
Milli q water, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Protein g sepharose 4 fast flow, by Cytiva (3 mentions)
Amicon ultra 4 centrifugal filter, by Merck Group (3 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (3 mentions)
Pei max, by Polysciences (3 mentions)
Kogenate fs, by Bayer (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (2 mentions)
Polybrene, by Merck Group (2 mentions)
Rifampicin, by Merck Group (2 mentions)
Accuri c6, by BD (1 mentions)
Fugene hd, by Promega (1 mentions)
Facsaria, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Sepharose cl 2b, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chromabond, by Macherey-Nagel (1 mentions)
Hepes sodium salt, by Merck Group (1 mentions)
Bio beads sm 2, by Bio-Rad (1 mentions)
38 protocols using millex gp filter
1
Isolation and Characterization of Environmental Bacteriophages
2
Lentiviral Knockdown of RGC32 in Lymphoblastoid Cells
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Lentiviral particles were generated using the GLTR system (34 (link)) by transfecting HEK-293 FT cells (gift from Dr H. Hochegger) with pLK01 scrambled shRNA (gift from Dr H. Hochegger), pLK01 shRNA RGC32.1, or pLK01 shRNA RGC32.2 and packaging vectors pVSV-G and psPAX 2 (gift from Dr H. Hochegger) using Fugene HD (Promega) in 10 cm dishes. Lentiviruses were harvested by pelleting cells, decanting the culture supernatant and filtering it through a 0.45 μm MillexGP filter (Millipore). The cleared supernatants were aliquoted and 1 ml of lentivirus was added to 5 × 106 GM12878 or IB4 cells. Cells were assayed for the constitutive expression of GFP after 5 days by flow cytometry using a BD FACS Canto or BD Accuri C6 (BD Biosciences). shRNA expression was induced by addition of doxycycline at 0.5 μg/ml to 2 × 105 cells in a 24-well plate. Cells were split after 5 days and then every 2 days. Cells were harvested at different time periods after shRNA induction and the number of GFP positive cells determined by flow cytometry as described above. Sorting of GFP-positive cells was performed using a BDFACSAria (BD Biosciences).
3
Recombinant FVIII Purification and Characterization
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Recombinant hFVIII-FL (Kogenate FS; gift from Bayer Corporation, Berkeley, CA, USA). B-domain deleted human FVIII 23 (link) and porcine FVIII (OL) were expressed in BHK-derived cells and purified as previously described 19 (link). All proteins were buffer exchanged and concentrated against 20 mM HEPES buffer at pH 7.4 containing 150 mm NaCl and 5 mM CaCl2 through 0.22 μm Millex® GP filter (Millipore, Carrigtwohill, Co. Cork, Ireland) to 2.7 mg mL−1 for the hFVIII-FL and pFVIII-BDD and to 0.7 mg mL−1 for the hFVIII-BDD. The protein concentration was monitored with Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Sci Inc, Waltham, MA, USA) and calculated based on the molar absorption coefficient at 280 and 320 nm 24 (link). Samples were prepared in identical solution conditions and protein to lipid ratios for each of the Cryo-EM, CD, DLS and thrombin generation experiments.
4
Gravity-driven Size-Exclusion Chromatography
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The gravity size-exclusion chromatography column (SEC) was prepared using 15 mL of sepharose CL-2B (GE Healthcare, Cytiva, Uppsala, Sweden) particle size 60–200 μm, pore size 100,000–20,000,000 Da, and 2% cross-linked agarose gel filtration matrix. A total of 15 mL of the sepharose CL-2B ethanol suspension were equilibrated with phosphate-buffered saline (PBS; pH 7.4), which was previously degassed and filtered with a 0.22 µm filter (Millex-GP filter, Millipore, Burlington, MA, USA). Then, 10 mL of the sepharose CL-2B was packed in a 15 mL Chromabond column with a polyethylene (PE) frit integrated at the bottom (Chromabond, Macherey-Nagel, Düren, Germany) and another PE frit at the top to allow the loading of the sample uniformly. The dimensions of the column used in this work were 1.5 cm diameter and 5.6 cm height. The void volume was 2.5 mL determined using dextran blue. Bovine serum albumin (BSA, Sigma, Burbank, CA, USA) was used to check the elution profile of the SEC column. Another column of 5 mL volume was prepared similarly. After chromatography, the column was cleaned by 10 volumes of elution buffer followed by 1 volume 1% (v/v) Triton, 1 volume of 0.5 M NaOH, and 10 volumes of elution buffer before reuse.
5
Expression and Purification of Recombinant Porcine FVIII
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B-domain deleted porcine FVIII (rpFVIII OL, POL1212) was expressed in BHK cells provided by Professor Lollar’s laboratory (Emory University, Atlanta, GA) and purified following established protocols [32 (link),38 (link)]. The fractions containing rpFVIII were combined, buffer exchanged against HBS-Ca buffer and concentrated through a 0.22 µm Millex® GP filter (Millipore, Carrigtwohill, Co. Cork, Ireland). The protein concentration was estimated with a Nanodrop Spectrophotometer (ND-1000 ThermoFisher Sci, Waltham, MA) [32 (link)].
6
Purification of MERS-CoV 3CLpro Protease
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Following dialysis, the pH of the sample was manually adjusted to 5.5
using 1m solution of MES, pH 5.5, and any precipitated protein
was removed by filtering through a 0.22-μm pore size Millex-GP
filter (Millipore). The filtered sample was then loaded at a flow rate
of 2 ml/min onto an 8-ml Mono S 10/100 column (Amersham Biosciences)
equilibrated in Buffer D. The column was then washed with 5×
column volume (40 ml) of Buffer D at a flow rate of 2 ml/min. Protein
was eluted using a 25× column volume (200 ml) and a linear
gradient to 50% Buffer E (50 mm MES, pH 5.5, 1 m NaCl,
0.05 mm EDTA, 10% glycerol, and 5 mm BME). Fractions
(2 ml) were collected, and those containing MERS-CoV 3CLprowere pooled (22 ml) and concentrated to ∼5 mg/ml.
using 1
was removed by filtering through a 0.22-μm pore size Millex-GP
filter (Millipore). The filtered sample was then loaded at a flow rate
of 2 ml/min onto an 8-ml Mono S 10/100 column (Amersham Biosciences)
equilibrated in Buffer D. The column was then washed with 5×
column volume (40 ml) of Buffer D at a flow rate of 2 ml/min. Protein
was eluted using a 25× column volume (200 ml) and a linear
gradient to 50% Buffer E (50 m
0.05 m
(2 ml) were collected, and those containing MERS-CoV 3CLprowere pooled (22 ml) and concentrated to ∼5 mg/ml.
7
Membrane Protein Reconstitution Protocol
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HEPES sodium salt, sodium cholate (Na-cholate) and MSP1D1 scaffolding protein were obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Sodium chloride (NaCl) and calcium chloride (CaCl2) were obtained from J.T. Baker (Mallinckrodt Baker, Inc. Phillipsburg, NJ). Bio-Beads SM-2 were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Phospholipids-DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt) and GC (D-galactosyl-β-1,1' N-nervonoyl-D-erythro-sphingosine, d18:1/24:1(15Z)) were obtained from Avanti Polar Lipids, Inc (Alabaster, AL, USA). All buffer solutions – HBS (NaCl 150 mM, HEPES 20 mM, pH 7.4) and HBS-Ca2+ (NaCl 150 mM, HEPES 20 mM, CaCl2 5 mM, pH 7.4) were filtered through 0.22 µm Millex® GP filter (Millipore, Carrigtwohill, Co. Cork, Ireland).
8
Colistin and Doripenem Susceptibility in A. baumannii
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A. baumannii ATCC 19606 (American Type Culture Collection [ATCC], Manassas, USA) was susceptible to both colistin and doripenem with MICs of 1 mg/L for both antibiotics. The strain was grown in cation-adjusted Mueller-Hinton broth (MHB; Oxoid, Australia; 20–25 mg/L Ca2+ and 10–12.5 mg/L Mg2+). colistin (Sigma-Aldrich, Saint Louis, USA) and doripenem (Doribax, Shinogi Inc, Osaka, Japan) were prepared using Milli-Q water (Millipore Australia, North Ryde, New South Wales, Australia) prior to each experiment and sterilized by filtration with a 0.22-μm pore size Millex GP filter (Millipore, Bedford, MA).
9
Recombinant FVIII Protein Production
10
Mitomycin C-Induced Phage JL22 Isolation
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E. coli strain 22 was cultured to the exponential growth phase at 37°C in LB. The bacterial suspension was treated with 2.0 μM mitomycin C and incubated for 2 h at 37°C with shaking. The suspension was then centrifuged at 3,000 × g for 15 min to remove bacterial cell debris and filtered using a 0.22-μm Millex-GP filter (Millipore). The filtrate from the previous step was concentrated using a 100-kDa Amicon Ultra centrifugal filter unit (Millipore) to a final volume of about 1 mL. The phage suspensions obtained were stored at 4°C. The suspension containing phage JL22 was also checked by PCR for the five target genes to confirm the successful induction of JL22.
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