The cells were washed three times with PBS and collected in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with a protease inhibitor cocktail (Calbiochem, San Diego, USA). Protein concentration was determined by staining with Coomassie Blue (Beyotime Biotechnology, Shanghai, China). Cellular protein lysates were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to a 0.22 mm polyvinylidene fluoride membrane (Millipore) and probed with specific antibodies. Specific bands were detected by ECL chromogenic substrate and quantified by densitometry (Quantity One software, Bio‐Rad). The GAPDH antibody was used as a control. Anti–caspase‐3, cleaved caspase‐3, poly (ADP ribose) polymerase protein (PARP), cleaved PARP, cyclin D1, CDK2 and MCL‐1 (1:1000) were purchased from Cell Signalling Technology. GAPDH antibody was purchased from Proteintech. All antibodies are listed in Table S3 .
Coomassie blue
Manufactured by Beyotime
Sourced in China
Coomassie Blue is a protein stain used for the detection and quantification of proteins in various laboratory applications, such as gel electrophoresis and Western blotting. It is a blue dye that binds to the basic amino acid residues in proteins, resulting in a blue-colored complex that can be detected and measured.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh antibody, by Proteintech (1 mentions)
Skim milk, by ABclonal (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Hrp goat anti mouse igg h l, by ABclonal (1 mentions)
Skim milk, by Sangon (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
High glucose dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Ros staining solution, by Merck Group (1 mentions)
Mpp iodide, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Curcumin, by Merck Group (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
18 protocols using coomassie blue
1
Protein Quantification and Western Blot Analysis
2
Western Blot Protein Visualization Protocol
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Protein samples were denatured by boiling before electrophoresis in a 10% SDS polyacrylamide gel. Proteins were visualized by staining with either Coomassie blue (Beyotime) or transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The PVDF membranes were blocked for 2 h at room temperature with 5% skim milk (Sangon, Shanghai, China) in PBST (0.05% Tween in 0.01 M phosphate-buffered saline (PBS)). Blocked membranes were then incubated with primary mouse anti His-Tag mAbs (ABclonal, China) diluted in 2% skim milk in PBST for 2 h at 25 °C and washed three times with PBST. Membranes were then incubated with horseradish peroxidase (HRP)-Goat Anti-Mouse IgG (H + L) (ABclonal, China) secondary antibodies in 2% skim milk in PBST for 2 h at 25 °C and were washed extensively. The signals were detected using enhanced chemiluminescence (ECL) reagents (Thermo, USA).
3
Curcumin-mediated Neuroprotection in PD
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Curcumin (purity ≥ 65%), MPP+ iodide, and ROS staining solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Soybean PC (l -α-lecithin) was procured from Ruixi Biotech Co. Ltd. (Xi’an, China). Cholesterol was procured from Avanti (Alabaster, AL, USA). Penicillin–streptomycin was obtained from Hyclone (USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM), trypsin EDTA, and fetal bovine serum were obtained from Gibco Life Technologies (AG, USA). The 4,6-diamidino-2-phenylindole (DAPI), annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit, Coomassie blue, radioimmunoprecipitation assay lysis buffer, bicinchoninic acid protein kit (BCA), and ROS assay kits were procured from Beyotime Biotechnology (China). MPTP-HCl and α-syn enzyme-linked immunosorbent assay kit were obtained from Lianshuo Chuangxiansheng Biotechnology Co. Ltd. (China). Monoclonal RAET1H antibody and monoclonal MICA/B antibody were purchased from Signalway Antibody (CA, USA). The NK cell isolation kit was obtained from Miltenyi Biotec (German). Rabbit anti-Lyve-1 antibody, mouse anti-TLR4 antibody, mouse anti-TH antibody, mouse anti-GFAP antibody, and rabbit anti-α-syn antibody were purchased from Servicebio (China).
4
SDS-PAGE Cytomembrane Characterization
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The AGS cytomembrane and ZTC@M with the same concentration were loaded onto the 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gels) for separation. Coomassie Blue (Beyotime, China) was used to stain the gel, and the picture was taken after destaining the gel with water for 6 h.
5
Kae Modulates Stability of HR1 and HR2 Peptides
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To test the effect of Kae on the stability of HR1 or HR2, HRs were incubated with isopropanol or various concentrations of Kae for 1 h at 37˚C, at final concentrations of 100 and 40 µM respectively. As for the formation of 6-HB, HR1 or HR2 were incubated with isopropanol or 100, 200 and 300 µM of Kae for 1 h at 37 ˚C. And HR2 or HR1 were then added to the mixture and incubated for an additional 30 min at 37˚C. The final concentrations of HR1 and HR2 were 100 µM and 40 µM respectively. Additionally, to determine the thermal stability of the original and mutant HR2 peptides, the samples were incubated at 37˚C for 0, 10, 20, 30, 40, 50 and 60 min. The original and mutant HR2 peptides concentrations were 40 µM. After the treatment, the samples were diluted with non-denaturing buffer and loaded onto a Bis-Tris gel (4-20%) for 2 h with a constant voltage of 125V, in an ice-water bath for the 6-HB formation assay and HR2 stability assay. For the effect of Kae on the stability of HR1, the diluted samples were applied to a Bis-Tris gel (10%) for 5 h at a constant voltage of 125V in an ice-water bath using reversing electrodes. The gel was then stained with Coomassie blue (Beyotime) and imaged using a ChemiDoc MP Imaging System.
6
Recombinant Production of chIL-9Rα Extracellular Domain
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chIl-9Rα gene with only extracellular domain was cloned into pET-28a plasmid using a pair of primers (Forward:5′-CGGGATCC (BamHI)AGAGATTTCCCTGGCAGT-3′ and Reverse:5′-CCGCTCGAG (XhoI)CGTTCTGGTGTCAAAGAG-3′) and transformed into E. coli ROSETTA (DE3) strain for recombinant expression by inducing with 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG ) (Solarbio, Beijing, China). After induction for 4 h, the lysates of bacteria were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE ) and stained with Coomassie Blue (Beyotime, Shanghai, China). In order to produce adequate rchIL-9Rα, 300 mL bacteria culture was induced at the above conditions. The bacteria pellets were sonicated, and the lysed precipitate were dissolved with 8 M urea. The rchIL-9Rα with his tag was purified with the Ni Sepharose Column (General Electric, Boston, MA) according to the manufacturer's protocol. The purified rchIL-9Rα was refolded by dialysis with gradient urea buffer (6 M, 4 M, 2 M, 1 M, 0 M) at 4°C, and finally quantified by BCA Protein Assay Kit (Beyotime, Shanghai, China).
7
Quantifying RVFV Gn Head-PA Fusion Protein Binding
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The maximum amount of RVFV Gn head-PA fusion protein bound to 1U GEM particles was determined by SDS-PAGE. Briefly, 1U of GEM particles was combined with a range of supernatant volumes (1, 2, 3, 4, 6, 8, and 10 mL) containing the RVFV Gn head-PA fusion protein. After binding for 1 h, the complexes were washed three times with PBS. At the same time, a BSA standard was twofold serially diluted. Samples were denatured with 5 × protein loading buffer at 95°C for 10 min. Equal volumes of samples were loaded on the gel, and the samples were separated by 12% SDS-polyacrylamide gel. After Coomassie blue (Beyotime Biotechnology, China) staining for 20 min followed by 10 washes, the maximum amount of RVFV Gn head-PA fusion protein that bound to 1U GEM particles was determined based on the BSA standard using the Quantity One image analysis software.
8
Membrane Protein Detection in Cell Vesicles
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The presence of membrane-associated proteins on RBC CM, LO2 CM, 4T1 CM, and A549/T CM (cell membrane vesicles) as well as on CM-HNPs was determined by Coomassie blue staining assay. The RBC CM, LO2 CM, 4T1 CM, A549/T CM, and CM-HNPs were collected after 0, 24, and 48 h. Next, the prehomogenated cell membrane vesicles and respective CM-HNPs were lysed in radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor solution on ice for 5 min. Next, lysates were centrifuged at 13,000× g for 5 min at 4 °C to collect the supernatants and subjected to BCA protein assay (Beyotime Biotechnology, Haimen, China) for total protein quantification. Thereafter, the individual supernatant was mixed with SDS loading buffer and heated up to 95 °C for 5 min. A 20 μg equivalent protein for each sample was added in each well of 10% SDS-PAGE in an electrophoresis chamber system (Bio-Rad Laboratories, Philadelphia, PA, USA) and run at 120 V for 1.5 h. Finally, the gel was stained with Coomassie blue (Beyotime Biotechnology, Haimen, China) overnight, followed by distaining, and the images were recorded.
9
Nanomedicine Cytomembrane Coating Validation
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Gel electrophoresis was used to verify that the nanomedicine was coated by cytomembrane. Different protein samples with the same concentration were loaded onto 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gels) for separation. The gel was then stained with Coomassie Blue (Beyotime, China) for 2 h and washed thoroughly with water for 6 h. The gel was observed under a microscope using ECL western blotting substrate and images were obtained.
10
Western Blot Analysis of Protein Expression
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Cells were washed three times with PBS and collected in RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Calbiochem, United States). Protein concentration was determined by staining with Coomassie Blue (Beyotime Biotechnology, China). After electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (Merck Millipore, Germany). After blocking with 0.1% Tween 20 (TBS-T) in Tris-buffered saline containing 5% skim milk for 1 h at room temperature, the primary monoclonal antibody, including anti-POU2F1 (#10387-1-AP, 1:500; Ptgcn), anti-E-cad (#20874-1-AP, 1:1000; Ptgcn), anti-Vimentin (#60330-1-Ig, 1:1000; Ptgcn), and anti-β-actin (#66009-1-Ig, 1:2000; Ptgcn), was added to the membrane and incubated overnight at 4 °C. The next day, the membrane was incubated with secondary antibody Goat Anti Rabbit IgG/HRP for 1 h at room temperature and the signal was detected in a Bio-Rad ChemiDoc XRS imaging system. The ratio of the gray value of the target protein to the gray value of β-actin indicates the relative amount of protein.
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