To test the protein level of tight junction protein (ZO-1, occludin and claudin-1), the phosphorylation level of the proteins, which belongs to the downstream of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway, were tested. The tissue samples were homogenized by using Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail (Songon Biotech, Shanghai, China). The concentration of protein was determined by using the BCA protein assay kit. The tissue sample was mixed with 5 × loading buffer (Sangon Biotech, Shanghai China) and heating at 100∘C for 5 min. The SDS-PAGE gel kits were purchased from Sangon Biotech (Shanghai, China). The sample (50 μg protein) was loaded to each well. The detailed process was described in the previous study (Zhai et al., 2018 (link)). The primary antibodies including β-actin, p38, p-p38, c-Jun, p-c-Jun, p65, p-p65, and ZO-1, claudin-1 were purchased from Cell signaling Technology (CST, Danvers, MA, United States) and occludin was purchased from Thermo Fisher Scientific (Waltham, MA, United States).
Loading buffer
Manufactured by Sangon
Sourced in China
Loading buffer is a solution used in gel electrophoresis to prepare DNA, RNA, or protein samples for loading into a gel. It helps to increase the density of the sample and track the sample movement during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 system, by BD (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
P c jun, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mini gel system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Sangon (1 mentions)
Antibody against nrf2, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Sangon (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Bca kit, by Sangon (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Temed, by Sangon (1 mentions)
Nonfat dried milk, by Sangon (1 mentions)
Annexin 5 fitc pi kit, by Sangon (1 mentions)
Monodansylcadaverine mdc kit, by Solarbio (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Ssdnas, by Sangon (1 mentions)
13 protocols using loading buffer
1
Protein Expression and Phosphorylation Analysis
2
Molecular Mass Determination of ds-Diabody
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The molecular mass of ds-Diabody was determined by reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Each sample was mixed with 5× loading buffer (Sangon, Shanghai, China) and heated for 10 min at 100°C. Samples were then separated under reducing condition on 12% (m/v) tris-glycine gels (Sangon) using a Mini-gel system (Bio-Rad). The proteins in the gel were stained with Coomassie (Sangon) and analyzed using the Automatic Analysis System of Electrophoresis Gel Imaging (Tanon Science Technology Co. Ltd, Shanghai, China).
3
Seleno-Aminopolysaccharide Antioxidant Pathway
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DAO, D-LA, and LPS kits were obtained from Nanjing Senbeijia Biological Technology Co., Ltd. (Nanjing, China). NO, TNOS, SOD, GSH-Px, CAT, and MDA were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2000 DNA Marker, RNA loading buffer, DNA 6×loading buffer, RIPA Lysis buffer, PMSF, BCA kit, BeyoECL Star kit, 5×loading buffer, BSA, non-fat dried milk, TEMED were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Monoclonal antibodies against β-actin were from Cell Signaling Technology, Inc. (Boston, MA, USA). Antibody against Nrf2 was acquired from Abcam (Cambridge, UK). The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Alcian Blue, periodic acid schiff were purchased from Beijing solab technology co., Ltd. (Beijing, China). All other reagents are of the highest grade or analytical grade. Our laboratory synthesized the low molecular seleno-aminopolysaccharide (LSA).
4
Apoptosis and Autophagy Assay Protocol
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Cells after treatment were collected and washed in PBS 3 times. They were resuspended in loading buffer (Sangon Biotech, Shanghai, China) to 2 × 105 to 5 × 105 cells/mL. For apoptosis and autophagy analysis, Annexin V-FITC/PI kit (Sangon Biotech) and monodansylcadaverine (MDC) kit (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) were utilized, respectively. BD FACSCanto™ II system (BD Biosciences, San Jose, CA, USA) was utilized to detect cell surface markers. All the results were analyzed by the FlowJo software.
5
Chondrocyte Apoptosis Analysis
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The chondrocytes were harvested and washed three times with phosphate-buffered saline (PBS) and resuspended in loading buffer (Sangon Biotech, Shanghai, China) to 2–5 × 105 cells/mL. For the apoptosis analysis, the Annexin V-fluorescein isothiocyanate/propidium iodide kit (Sangon Biotech, Shanghai, China) was used strictly according to the manufacturer’s protocol. The Becton, Dickinson (BD) FACSCanto™ II system (BD Biosciences, CO, USA) was used to detect the surface markers of the chondrocytes. The results were analyzed using the FlowJo software.
6
ssDNA Cleavage by PfAgo Nuclease
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A 59 bp stretch of ssDNA was synthesized in the middle of the PRA amplified sequence, and 8 complementary gDNA were designed every 4 bases. gDNAs were prepared by phosphorylating the synthesized 16 bp ssDNA using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA). The 25 µL PfAgo cleavage ssDNA reaction contained 2.5 µL of 10 × reaction buffer (150 mM Tris/HCl, 1 M NaCl, pH 8.0), 0.2 µM PfAgo (JiaoHong Biotech, Shanghai, China), 0.8 µM ssDNA, 0.6 µM gDNA and 2 µM MnCl2; additionally, ddH2O was added to 25 µL. After 1 h of incubation at 95°C, the product was combined with a 2 × loading buffer (Sangon, Shanghai, China) mixture and subsequently analyzed with 16% urea-denaturing PAGE in 1 × Tris−borate−ethylenediaminetetraacetic acid (TBE ).
7
Flow Cytometry Analysis of CD163 Expression
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After cells were treated, they were collected and washed in PBS three times. The cells were suspended in loading buffer (Sangon Biotech Co., Ltd.,) to a density of 2–5×105 cells/ml, then incubated with a phycoerythrin (PE)-conjugated mouse anti-CD163 monoclonal antibody (1:100; cat. no. sc-20066; Santa Cruz Biotechnology) in the dark for 30 min at 37°C. After removal of the antibody solution, the cells were washed with PBS twice and then resuspended in loading buffer. A BD FACSCanto™ II flow cytometer (BD Biosciences) was used to detect surface markers of the cells. The results were analyzed by FlowJo software v.10.0.7 (FlowJo LLC).
8
Magnetic Bead-Based CRISPR Detection
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Monodispersed Magnetic Streptavidin Microspheres (MBs) were purchased from Tianjin Bessel Chromatography Technology Development Center (Tianjin, China), with a solid content of 0.5% (w/v) and a particle size of 300 nm. ZEN, Ochratoxin a (OTA), Fumonisin (FB1), Aflatoxin B1 (AFB1) and Aflatoxin M1 (AFM1) were bought form Meizheng Testing Technology Co., Ltd. (Beijing, China). EnGen LbaCas12a from Lachnospiraceae bacterium ND2006 was purchased from New England Biolabs (Ipswich, MA, USA). CRISPR RNA (crRNA) and other ssDNAs were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Sequence details are given in Table S1 . The buffer solution 1 was used as a reaction solution for the streptavidin-modified magnetic beads and DNA. The CutSmart Buffer and NEBuffer™ 2.1 was from New England Biolabs. Buffer components are visible in Table S2 . PBS, agarose, 50 × TAE, 5 × TBE, EDTA, 30% acrylamide solution (ACR-BIS, 29:1), tetramethylethylenediamine (TEMED), PAGE coagulant, ammonium persulfate (APS)+, 6× loading buffer and DNA Marker were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). RNase-free water was employed as the solvent throughout the experiment. All chemical reagents were of analytical grade.
9
Screening of Novel Cancer Therapeutics
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All oligonucleotides were obtained from Sangon Biotechnology Co., Ltd (Shanghai, China). All oligonucleotides and miRNAs were purified with HPLC. The sequence of oligos used is detailed in Table S1.† DEPC treated deionized water, 40% acrylamide, 6× loading buffer and 4S GelRed dye were purchased from Sangon Biotechnology Co., Ltd (Shanghai, China). Penicillin-streptomycin, 4-(2-hydroxyethyl) piperazine-1-ethylate sodium salt (HEPES), magnesium chloride, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) were purchased from Sigma-Aldrich (MO, USA), and all were analytically pure reagents. Dulbecco's modified Eagle Culture medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Thermo Fisher Scientific (USA). MCF-7 cells, MDA-MB-231 cells, A549 cells, MCF-10A cells and MRC-5 cells were purchased from Shanghai Institute for Biological Sciences (SIBS).
Gel electrophoresis was performed using a DyCZ-24DN electrophoresis cell (LIUYI, Beijing, China) and DigiGenius gel system (Syngene, UK). Fluorescence spectra were measured using a WATERS Prep 150 fluorescence spectrophotometer (WATERS, USA). Confocal fluorescence images of cells were obtained using a LSM 880 confocal laser scanning fluorescence microscope (Carl Zeiss, Germany). Cell viability was determined using a M1000PRO Multifunctional Microplate Reader (TECAN, Austria).
Gel electrophoresis was performed using a DyCZ-24DN electrophoresis cell (LIUYI, Beijing, China) and DigiGenius gel system (Syngene, UK). Fluorescence spectra were measured using a WATERS Prep 150 fluorescence spectrophotometer (WATERS, USA). Confocal fluorescence images of cells were obtained using a LSM 880 confocal laser scanning fluorescence microscope (Carl Zeiss, Germany). Cell viability was determined using a M1000PRO Multifunctional Microplate Reader (TECAN, Austria).
10
Aptamer Stability Assay in Serum
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About 250 nM FAM-labeled aptamers were incubated with RPMI 1640 with 10% fetal bovine serum (FBS) at 37°C. At designated time points, samples were heated at 95°C for 10 min to denature the enzyme and subsequently stored at −20°C until all samples were collected. Ten microliters DNA samples were mixed with 2 μl 6 × loading buffer (Sangon) and then loaded into 10% polyacrylamide gel in electrophoresis buffer (9 mM Tris, pH 8.0, containing 9 mM boric acid and 1 mM EDTA). After electrophoresis, the gels were analyzed with a molecular imager (GE Healthcare).
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