0.2% gelatin (Solarbio, Beijing, China) was incubated at 37 °C for 1 h in the well containing a coverslip and cross-linked with 1% glutaraldehyde (Aladdin, Shanghai, China). The excess glutaraldehyde was permeabilized with 1 M glycine (Biofroxx, Guangzhou, China) for 20 min. Then, HFF cells (5 × 104) were seeded into gelatin-coated well and cultured in complete medium supplemented with 50 µg/mL ascorbic acid (Solarbio, Beijing, China) for 7–10 days. Subsequently, HFF cells were removed using extraction buffer (1 mL NH4OH, 250 µL 0.5 % Triton-X-100, 48.75 mL PBS). The remaining cell were removed with 20 μg/mL DNAse I (Solarbio, Beijing, China) [36] (link), [37] (link). Breast cancer cells treated with BD or transfected with plasmids or siRNAs were seeded onto the extracted ECM, and incubated for 72 h at 37 °C, followed by immunofluorescence staining of Fibronectin 1 (FN1), COL1A2 and DAPI, and photographing with a laser confocal fluorescence microscope.
Glycine
Manufactured by neoFroxx
Sourced in China, Germany
Glycine is a colorless, crystalline amino acid that serves as a fundamental building block for proteins. It is an essential component in various biochemical processes within living organisms.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Solarbio (1 mentions)
Ascorbic acid, by Solarbio (1 mentions)
Tris hcl, by Avantor (1 mentions)
Poly ethylene glycol, by Yuanye Bio-Technology (1 mentions)
Polyvinylpyrrolidone, by Yuanye Bio-Technology (1 mentions)
L cysteine, by Merck Group (1 mentions)
Sds page gel kit, by Solarbio (1 mentions)
Apollo staining solution, by RiboBio (1 mentions)
Hoechst 33342 reaction solution, by RiboBio (1 mentions)
Tritonx 100, by RiboBio (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Formaldehyde, by Biosharp (1 mentions)
Protein a g conjugated agarose beads, by Smart-Lifesciences (1 mentions)
Proteinase k, by Tiangen Biotech (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Bioruptor plus, by Diagenode (1 mentions)
Phosphorylase inhibitor, by Beyotime (1 mentions)
Protein loading buffer, by Beyotime (1 mentions)
Sds page gel preparation kit, by Biosharp (1 mentions)
Bca protein quantitative detection kit, by Beyotime (1 mentions)
6 protocols using glycine
1
Decellularized Extracellular Matrix Preparation
2
Prunus mume Fruit Pulp Extraction
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Prunus mume Sieb. et Zucc. were purchased at commercial maturity in Dazhou, Sichuan province in June. The pulps were cut from the fruits at 4 °C and frozen at −78 °C until used. Polyvinylpyrrolidone (PVPP, USP), catechol, polyethylene glycol (PEG 15000), and cellulose membranes (76 × 49 mm) were purchased from Yuanye (Shanghai, China). l -Cysteine (Biological reagent, BR) was purchased from Sigma-Aldrich (USA). Tris–HCl was purchased from Amresco (USA). 4-Hexylresorcinol (BR) was purchased from Aladdin (USA). SDS-PAGE Gel Kit and Tris were purchased from Solarbio (Beijing, China). Glycine (BR) was purchased from Biofroxx (Germany). Markers were purchased from Tiangen (China). Bovine albumin serum (BSA, BR) was purchased from Regal (Shanghai, China). All other reagents were purchased from KESHI (Chengdu, China), including the brilliant blue G250 (BR). All reagents and chemicals used in this study were of analytical grade, except where indicated otherwise.
3
Quantifying Cell Proliferation Using EdU Assay
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Cell proliferation assays were performed using the Cell-LightTM EdU Apollo In Vitro Kit (RiboBio, Guangzhou, China). Cancer cells (1 × 105) were seeded in 96-well plates and treated according to the experimental requirements. Subsequently, EdU medium was added and the cells were incubated for 2 h. After washing the cells twice with PBS, they were fixed with 4% PFA solution (Biosharp) for 30 min. Cells were then incubated with glycine (2 mg/ml; Biofroxx, Germany) for 5 min and permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min. After adding 1 × Apollo® staining solution (RiboBio) and incubating for 30 min at light avoidance and RT, the cells were washed three times with 0.5% TritonX-100. Finally, a 1 × Hoechst 33342 reaction solution (RiboBio) was added and incubated for 30 min with light avoidance at RT. The cells were imaged using a microscope (Leica Microsystems) and counted in four randomly selected fields.
4
ChIP Assay for Protein-DNA Interactions
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The ChIP assay was performed as described previously (28 (link), 29 (link)). Briefly, 1 × 107/mL cells were fixed in 1% formaldehyde (Biosharp, Hefei, China) for 20 min at room temperature, and 125 mM glycine (BioFroxx, China) was added to quench formaldehyde. After a 30-min incubation in ChIP lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor) on ice, the lysates were sheared by sonication using a Bioruptor Plus (Diagenode, Liège, Belgium). Cross-linked chromatin samples were incubated with the indicated antibodies or normal rabbit IgG in a rotator at 4°C overnight. Subsequently, protein A/G-conjugated agarose beads (Smart-Lifesciences, Changzhou, China) were added, and the samples were incubated overnight at 4°C in a rotator, collected, and washed three times. To elute DNA fragments, immunocomplexes were incubated with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1.0% SDS) for 2 h at 65°C. One half of the eluted immunocomplexes was saved as the immunoprecipitation sample, while the other half was treated with proteinase K (Tiangen Biotech) overnight at 55°C. Finally, the DNA was purified with a TIANamp Genomic DNA kit (Tiangen Biotech). The purified DNA was detected by qPCR. The antibodies used for ChIP and the primers used for qPCR are listed in Tables 3 and 4 .
5
Virus Extraction and Concentration Protocol
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Before the experiment, the equipment is wiped and operating table surface with 0.5% sodium hypochlorite solution (Abd-Elmaksoud et al., 2014 (link)), and then wipe with water after 15 min. Use a peristaltic pump (YZ1515X; Runze, Shenzhen City, China) to extract the seeded virus water from the container and filter it through a glass wool filter element at different speeds, and let the peristaltic pump continue to work for 3 min after all the water has been filtered. Soak the glass wool filter element with 75 ml of 3% beef extract buffer (B8570; Solarbio, Beijing, China) solution containing 0.5 M glycine (1275KG2P5; BioFroxx, Hangzhou, China) and pH 9.0 for 20 min, then wash with 75 ml of beef extract buffer solution again. Collect the total 150 ml buffer solution in a clean container, adjust the eluent pH to neutral with 1M hydrochloric acid, and store at 4 °C; if over 48 h, it must be stored at −20 °C or lower temperature.
6
RIPA Lysis and Protein Quantification
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RIPA lysate (P0013B Beyotime Biotechnology), PMSF (100 mM) (BL507A biosharp), Phosphorylase inhibitor (P1081 Beyotime Biotechnology), BCA protein quantitative detection kit (P0012 Beyotime Biotechnology), 5 × protein loading buffer (P0015 Beyotime Biotechnology), SDS–PAGE Gel preparation Kit (BL508A biosharp), Protein Marker (20350ES72 YEASEN), TRIS(1115GR500BIOFROXX), Glycine (1275KG2P5 BIOFROXX).
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