Tris-buffered saline (TBS) was prepared by mixing 6.1 g of Trizma base (Sigma, Darmstadt, Germany) with 9.0 g of sodium chloride (Sigma) in 800 mL of distilled water. The pH was adjusted to 7.6 using concentrated hydrochloric acid (Sigma-Aldrich, Darmstadt, Germany) and the solution made up to 1 L. TBS/Tween 20 1% solution was prepared by mixing 5 mL of Tween 20 (Becton Dickinson, NJ, USA) with 500 mL of TBS.
Tween 20
Manufactured by BD
Sourced in United States
Tween 20 is a non-ionic detergent commonly used as a laboratory reagent. It is a polysorbate compound primarily used as a surfactant in various applications, including cell lysis, protein extraction, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Trizma base, by Merck Group (1 mentions)
Anti vegf antibody, by Santa Cruz Biotechnology (1 mentions)
T5168, by Merck Group (1 mentions)
Olive oil, by Loba Chemie (1 mentions)
Clove oil, by Loba Chemie (1 mentions)
Tween 80, by BD (1 mentions)
Castor oil, by Loba Chemie (1 mentions)
Isopropyl alcohol, by Merck Group (1 mentions)
Nutrient broth, by BD (1 mentions)
Bacto agar, by BD (1 mentions)
Difco nutrient broth, by BD (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Goat serum, by BD (1 mentions)
Fluorescent mounting media, by Agilent Technologies (1 mentions)
Donkey anti rat alexa647, by Thermo Fisher Scientific (1 mentions)
7 protocols using tween 20
1
Preparing Tris-Buffered Saline with Tween 20
2
VEGF Protein Expression Analysis in U-87 Cells
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U-87 cells were lysed using RIPA buffer (cat. no. 9806; Cell Signaling Technology, Inc.) supplemented with a protease inhibitor mixture (P3100; GenDEPOT) and a phosphatase inhibitor mixture (P3200; GenDEPOT). Total protein content was determined by Pierce™ BCA Protein Assay Kit (cat. no. #23225; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total protein (20–30 µg/lane) was separated on 12% gels using SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham; Cytiva). After transfer, the membrane was incubated with 5% skimmed milk (cat. no. #232100; Becton, Dickinson and Company) in PBS containing 0.1% Tween-20 (cat. no. TR1027-500-00; Biosesang) for 1 h at room temperature. The membrane was immunoblotted using an anti-VEGF antibody (1:1,000; sc-7269; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C. An α-tubulin antibody (1:3,000; T5168; Sigma-Aldrich; Merck KGaA), as the internal control, was incubated for 16 h at 4°C. For the incubation with secondary antibody, HRP-conjugated anti-mouse antibody (1:10,000; #1031-05; SouthernBiotech) was applied for 1 h at room temperature. Chemiluminescence intensity was measured using an ImageQuant™ LAS 4000 apparatus (Cytiva). The quantification of band intensity was performed using ImageJ software (V1.8.0; National Institutes of Health) and normalized to the intensity of α-tubulin.
3
Formulation and Characterization of Topical Oils
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TF-HCl was a generous donation from Mass Pharma Pvt. Ltd Lahore (Pakistan). Castor oil, clove oil, olive oil, and coconut oil were purchased from Loba Chemie (India). Ethanol and isopropyl alcohol were from Merck (Germany) and Tween 80 and Tween 20 from BDH (India). All the chemicals used were of analytical grade. The purified water was obtained from RO system Jawa Pharmaceuticals Pvt. Ltd Lahore (Pakistan) and distillation plant (Riphah Institute of Pharmaceutical Sciences, Riphah International University, Lahore, Pakistan).
4
Micrococcus luteus Growth and Agar Preparation
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Micrococcus luteus (ATCC 10240) was grown in Difco nutrient broth (Becton Dickinson and Co., Sparks, MD) at 30°C in an orbital shaker at 150 rpm for 24 h and refrigerated at 4°C before use. The agar media for this assay consisted of 0.8% nutrient broth, 0.75% Bacto agar (Becton Dickinson and Co.), and 1% Tween 20 (BDH®, Solon, OH).
5
Immunohistochemical Staining of Murine Brain Vasculature
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Brains were prepared 48 h post adoptive cell transfer. Brains were embedded in Tissue-Tek (Sakura) immediately after explantation and cut into 100 µm thick coronal tissue slices and stored at -80°C. Three sections per brain in 500 µm distance were stained and analyzed as follows. Sections were dried at RT for 30 min, fixed in ethanol-acetone (1:1, Sigma) for 10 min at RT, washed 3 times 5 min in PBS (PAN Biotech), blocked in PBS (PAN Biotech) + BSA (1%; PAN Biotech) + goat serum (10%; Sigma)+ tween20 (0.2%; Sigma) for 50 min at RT. Then sections were stained for 120 min at RT in PBS + BSA + goat serum + tween20 + primary antibody (rat anti CD31, BD; 1:100), washed 3 times 5 min in PBS and stained in PBS + 1% BSA + 10% goat serum + 0.2% tween20 + secondary antibody (donkey anti rat-Alexa647, life technologies; 1:100) for 60 min at RT in the dark. After washing 3 times in PBS (PAN Biotech), DAPI staining (1 µg/ml; Sigma) was performed for 10 at RT and the sections were embedded in fluorescent mounting media (Dako).
6
Immunohistochemical Evaluation of Human Cells
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The presence of reminiscent human cells was evaluated by fluorescent immunohistochemistry staining targeting human leukocyte antigen (HLA) complexes of human discogenic cells. Sections were deparaffinized and incubated in 0.0005% proteinase (Sigma‐Aldrich, St. Louis, Missouri, USA) tris–HCl (pH 7.6) for 10 minutes at 37°C. Sections were rinsed in PBS and treated with 100 μL 3.0 IU/mL hyaluronidase type V (Sigma‐Aldrich), 0.05 IU/mL chondroitinase‐ABC (Sigma‐Aldrich) PBS for 60 minutes at 37°C. Subsequently, sections were rinsed and blocked with 3% bovine serum albumin (BSA), 0.2% Tween‐20 (Sigma‐Aldrich) PBS for 30 minutes at room temperature. Primary antibody antihuman HLA‐ABC Purified (BD BioScience, Frankilin Lakes, New Jersey, USA) was diluted 1:25 in 1% BSA, 0.2% Tween‐20 PBS, applied and incubated over night at 4°C. Subsequently, samples were rinsed, coated with secondary goat antimouse IgG/IgM Alexa Fluor 488 (Thermo Fisher), and incubated for 1 hour at room temperature. Sections were consecutively rinsed and mounted with VECTASHIELD HardSet Mounting Medium with 4′ 6‐diamidino‐2‐phenylindole (DAPI) (Vector Laboratories, Burligame, California, USA). Finally, all slides were analyzed by LSM 510 META confocal microscope (ZEISS, Oberkochen Germany).
7
Hemagglutinin Binding Kinetics Assay
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All binding steps were performed in PBS pH 7.2 containing 3% BSA (Sigma) and 0.05% Tween-20 (BDH). ScFv-Fc antibodies at 10 μg ml−1 were immobilized on Protein A biosensors (Forte Bio cat no 18-5010) for 300 s before an equilibration step for 180 s. Bound antibodies were associated with a minimum of 5 concentrations of biotinylated hemagglutinin H1 (A/California/07/2009 H1N1) or hemagglutinin H5 (A/Vietnam/1203/2004 H5N1) for 300 s, then dissociated in buffer for 600 s or 2000 s for high-affinity antibodies. The affinity of the 0084-B14 antibody was assayed as above, but using Anti-human Fc biosensors (Forte Bio cat no 18-5060). Positive binding was determined where the background-corrected delta value was greater than 0.03 nm. For KD determination, curves were fit to a standard 1:1 binding model using analysis software by Forte Bio. Only curves that showed good fits to this model (R2 > 0.96) were used in affinity calculations.
Competition assays were performed by immobilizing biotinylated antibody to streptavidin biosensors (Forte Bio cat no 18-5019) at 10 μg ml−1 for 1200 s before equilibrating the sensors in buffer containing 250 μM d-biotin (Amresco) for 180 s. Association was performed with 1 μM hemagglutinin H1 (A/California/07/2009 H1N1) or buffer for 500 s before associating with competing IgG for 500 s.
Competition assays were performed by immobilizing biotinylated antibody to streptavidin biosensors (Forte Bio cat no 18-5019) at 10 μg ml−1 for 1200 s before equilibrating the sensors in buffer containing 250 μM d-biotin (Amresco) for 180 s. Association was performed with 1 μM hemagglutinin H1 (A/California/07/2009 H1N1) or buffer for 500 s before associating with competing IgG for 500 s.
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