Polyethyleneimine (PEI), branched, 50%, 750,000 Da; m-phenylenediamine (mPD); p-phenylenediamine (pPD); glucose; 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); dimethyl sulfoxide (DMSO); bovine serum albumin (BSA); Tween® 20 and Whatman™ chromatography 1CHR paper; hemoglobin (Hb); glutaraldehyde, 25% (w/w) aqueous solution; and guaiacol and glucose oxidase (GOX) from Aspergillus niger were purchased from Sigma-Aldrich® (Madrid, Spain). Myeloperoxidase (MPO) from human leukocytes was purchased from Planta Natural Products (Vienna, Austria). PERDROGENTM 30% H2O2 (w/w) was obtained from Fluka (Madrid, Spain), phosphate buffered saline tablets were purchased from Fisher Bioreagents (Madrid, Spain), and rabbit polyclonal anti-myeloperoxidase antibody was obtained from Abcam (Cambridge, UK).
Tween 20
Manufactured by Cytiva
Sourced in United States
Tween-20 is a non-ionic detergent commonly used in various laboratory applications. It is a polysorbate surfactant that can be used to solubilize proteins, stabilize enzymes, and facilitate the mixing of hydrophobic and hydrophilic substances. Tween-20 is a versatile and widely-used reagent in biochemical and cell-based research.
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Lab products found in correlation
Whatman, by Cytiva (3 mentions)
Bovine serum albumin (bsa), by Cytiva (2 mentions)
Dimethyl sulfoxide (dmso), by Cytiva (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Ecltm western blotting detection reagent, by Cytiva (2 mentions)
Phosphate buffered saline tablets, by Thermo Fisher Scientific (1 mentions)
2 2 azino bis, by Cytiva (1 mentions)
Rabbit polyclonal anti myeloperoxidase antibody, by Abcam (1 mentions)
Amicon centrifugal filter unit, by Merck Group (1 mentions)
Whatman chromatography 1chr paper, by Cytiva (1 mentions)
Glucose oxidase gox from aspergillus niger, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Gold 3 chloride hydrate, by Merck Group (1 mentions)
Sodium citrate tribasic dehydrate, by Merck Group (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Pip strip, by Echelon Biosciences (1 mentions)
Bovine serum albumin bsa, by Bovogen (1 mentions)
Dulbecco s modified eagle s medium f12 dmem f12, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
8 protocols using tween 20
1
Enzymatic Peroxidase Colorimetric Assay
2
Optimized Paper-based Enzymatic Sensor
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Polyethyleneimine branched 50 % (PEI) 750000Da, m-phenylenediamine (mPD), pphenylenediamine (pPD), glucose, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), Tween ® 20 and Whatman TM chromatography 1CHR paper, Amicon® centrifugal filter units and glucose oxidase (GOX) from Aspergillus niger were purchased from Sigma-Aldrich ® . Human myeloperoxidase (MPO) was purchased from Planta Natural Products (PNP, Vienna Austria). A wound exudate sample from an infected wound was kindly provided by Dr….from Hospital of Terrassa.
In order to improve the fluidity of enzymes through the Whatman TM chromatography 1CHR paper, strips were cut (1.5 cm x 3 cm), soaked for 2 h at RT in a buffer solution (200 mM sodium phosphate buffer pH 6.5) containing 0.5 % BSA and 0.1 % Tween ® 20 to and dried overnight at 37 ºC. Stocks of compounds mPD and pPD (20 mM) were prepared in DMSO. A mixture containing one of the selected phenylenediamines (2 mM), ABTS (2 mM), glucose (2 mM) and PEI (10 %) prepared in 200 mM phosphate buffer pH 6.5 was applied by drop casting (2.5 l) on the centre of the paper strip and dried with air stream.
In order to improve the fluidity of enzymes through the Whatman TM chromatography 1CHR paper, strips were cut (1.5 cm x 3 cm), soaked for 2 h at RT in a buffer solution (200 mM sodium phosphate buffer pH 6.5) containing 0.5 % BSA and 0.1 % Tween ® 20 to and dried overnight at 37 ºC. Stocks of compounds mPD and pPD (20 mM) were prepared in DMSO. A mixture containing one of the selected phenylenediamines (2 mM), ABTS (2 mM), glucose (2 mM) and PEI (10 %) prepared in 200 mM phosphate buffer pH 6.5 was applied by drop casting (2.5 l) on the centre of the paper strip and dried with air stream.
3
Colorimetric Glucose Assay with PEG-AuNPs
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Materials D-(+)-Glucose (≥99.5%), 3,3′,5,5′-tetramethylbenzidine (TMB, ≥99%), horseradish peroxidase (HRP, Type VI-A, salt free), glucose oxidase (GOX) from Aspergillus niger (100 000-250 000 U g -1 , Type X-S), Tween 20, Whatman filter paper Grade 6 (3 µm pore, cellulose, ashless), Grade 1 (11 µm pore, cellulose, ashless), Grade 41 (20 µm pore, cellulose, ashless), poly (sodium 4-styrenesulfonate) (PSS, 30% in water), poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (PEG-COOH, M n 2100), sodium citrate tribasic dehydrate (≥99.0%) and gold(III) chloride hydrate (99.995%) were purchased from Sigma-Aldrich. Bovine Serum Albumin (BSA, protease free) was pur-chased from VWR. Phosphate buffered saline (PBS) containing 1 mg mL -1 BSA and 0.05% Tween was prepared following standard procedures. Polyethylene glycol coated gold nanoparticles (PEG-AuNPs, 20 nm) were fabricated as described elsewhere. 19
4
MINERVA Protein Binding Assay
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The PIP strips (Echelon Biosciences, Salt Lake City, UT, USA) were blocked with phosphate-buffered saline containing 0.5% (v/v) Tween-20 (Amersham Biosciences, UK) and 3% (w/v) bovine serum albumin (BSA; Bovogen, East Keilor, Australia) for 1 h and incubated with MINERVAFL or MINERVAΔC for 1 h. MINERVAFL bound strips were treated with α-MINERVA rabbit antibody (5122; Cell Signaling Technology, Danvers, MA, USA) for 1 h then with HRP-conjugated α-rabbit IgG goat antibody (A120-101P; Bethyl Laboratories, Montgomery, TX, USA) for 1 h. MINERVAΔC bound strips were treated with HRP-conjugated α-His6-tag antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The strips were washed with the blocking buffer for 5 min three times between each step. The strips were visualized with an ECL kit (Amersham Biosciences, UK).
5
Triclosan Cytotoxicity Signaling Pathway
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Triclosan was obtained from Sigma (St. Louis, MO, USA) while Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), B-27 supplement and FGF were purchased from Invitrogen (Carlsbad, CA, USA). EGF was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Penicillin/streptomycin, 0.25% trypsin-EDTA, Tween® 20, and ECLTM Western blotting detection reagents were obtained from Amersham Life Science (Arlington Heights, IL, USA).
Antibodies were purchased from the following companies: anti-β-actin from Sigma (St. Louis, MO, USA); caspase3, cleaved caspase 3, ERK, phosphor-ERK, JNK, phosphor-JNK, PI3K, phosphor-PI3K, p38, phosphor-p38, Akt and phosphor-Akt from cell signaling (Boston, MA, USA); Bax from BD PharmingenTM (BD biosciences, USA); Bcl-2 and cytochrome C from Santa Cruz (CA, USA).
Antibodies were purchased from the following companies: anti-β-actin from Sigma (St. Louis, MO, USA); caspase3, cleaved caspase 3, ERK, phosphor-ERK, JNK, phosphor-JNK, PI3K, phosphor-PI3K, p38, phosphor-p38, Akt and phosphor-Akt from cell signaling (Boston, MA, USA); Bax from BD PharmingenTM (BD biosciences, USA); Bcl-2 and cytochrome C from Santa Cruz (CA, USA).
6
Immunoblotting Protein Detection Protocol
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Proteins were run on SDS-PAGE gels and transferred to nitrocellulose membrane by electroblotting. Ponceau-S (0.1% Ponceau-S (w/v) (Sigma), in 5% acetic acid) staining was used to identify the location of the proteins on a PVDF membrane. The membrane was blocked with 5% milk powder or BSA, 0.1% Tween 20 (Sigma-Aldrich), for 15 h at 4 °C. Antiserum/antibody was diluted in PBS, 5% milk powder or BSA, 0.1% Tween-20, and protein bands were visualized using the enhanced chemiluminescence (ECL) substrate kit (Amersham) and X-ray film sheets.
7
Sterilization and Germination of Plant Seeds
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Seeds were washed with 100% ethanol for a second and sterilised with 2–10% (w/w) hypochlorite solution (Wako) containing 0.2% Tween-20 (Amersham Biosciences, Sweden) for 40 min. The sterilised seeds were washed with sterilised water and planted on Murashige and Skoog (MS) medium [MS salt (4.6 g l−1), 0.2% (w/v) gellan gum (Wako), 0.1% (v/v) Gamborg’s vitamin solution (Sigma-Aldrich Co. LLC, MO), pH 5.8]. Sterilisation time, gellan gum concentration and sucrose concentration differed depending on the purpose of the experiment. Details of these differences are described in the result section separately. To determine the appropriate germination conditions, three replications were conducted for each treatment with ca. 30 seeds per plate. Plates were incubated at 24 °C under a 16L: 8D cycle.
8
Mitochondrial Function and Senescence Assays
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The materials used in this study are as follows: Dulbecco’s modified Eagle medium (DMEM)/F12, Penicillin-Streptomycin (P/S), 0.25% trypsin-EDTA, and 10% Fetal Bovine Serum (FBS) were from Gibco BRL (Grand Island, NY, USA); Tween® 20 and ECLTM Western blotting detection reagent were from Amersham Life Science (Arlington Heights, IL, USA); anti-β Actin was obtained from Sigma (St. Louis, MO, USA); anti-iNOS and senescence detection kit were the product of Abcam (Cambridge, UK); minoxidil was obtained from Sigma; Agilent Seahorse XF Cell Mito Test Kit was from Agilent Technologies (CA, USA); Alexa Fluor® 594 conjugated Escherichia coli (K-12 strain) BioParticles® and Tetramethylrhodamine Methyl Ester (TMRM) were purchased from Thermo Fisher Scientific (MA, USA); MitoSOX™ was from Invitrogen (MA, USA).
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