PBMCs (1 x 107 cells) were lysed by 200 μl RIPA lysis buffer (CWBIO, Beijing) supplement with protease inhibitor cocktail (CWBIO, Beijing) and phosphatase inhibitor cocktail (CWBIO, Beijing). After lysis at 4°C for 15 min, the proteins were centrifuged at 8000 x g for 15 min. The protein concentration in the supernatant of cell extracts was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai). Then, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime, Guangdong) and transferred to polyvinylidene difluoride membranes (Millipore, American). The membranes were blocked with 5% no-fat powdered milk (Sangon, Shanghai) in tris-buffered saline (Solarbio, Beijing) with 1% tween (Solarbio, Beijing) for 2 h at room temperature. The membrane was probed with diluted primary antibodies CITED2 (Abclonal, 1:1,000), VEGF (Bioss, 1:1,000), and β-actin (Bioworld, 1:10,000) overnight at 4°C. On the next day, the membrane was re-probed with a secondary antibody, goat anti-rabbit antibody IgG (CWBIO, 1:20,000), labeled by enhanced chemiluminescence hypersensitive luminescent solution (Millipore, American) for 2 h at room temperature, and quantified by densitometry.
Tris buffered saline (tbs)
Manufactured by Solarbio
Sourced in China
Tris-buffered saline is a commonly used buffer solution that maintains a stable pH and ionic strength. It is composed of tris(hydroxymethyl)aminomethane (Tris) and sodium chloride (NaCl) in deionized water. This buffer solution is widely utilized in a variety of laboratory techniques and applications that require a physiologically relevant, neutral pH environment.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Solarbio (3 mentions)
Bca protein assay kit, by Solarbio (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Phosphatase inhibitor cocktail, by CWBIO (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Vascular endothelial growth factor (vegf), by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Skim milk, by Solarbio (1 mentions)
Ab6302, by Abcam (1 mentions)
Myc tag, by ABclonal (1 mentions)
Af5003, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
Rat insulin elisa kit, by Mercodia (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
9 protocols using tris buffered saline (tbs)
1
Western Blot Analysis of CITED2, VEGF in PBMCs
2
Western Blot Analysis of Protein Interactions
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Whole-cell lysate and the Co-IP supernatant were mixed with 5× SDS-PAGE Plus sample buffer (GenStar, Beijing, China) and then incubated in a boiling water bath before separation by SDS-PAGE and subsequent transfer to PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% skim milk (Solarbio, Beijing, China) for 3 h then incubated with primary antibody overnight at 4°C. The membrane was washed thrice for 10 min each time using Tris-buffered saline and 0.1% Tween-20 (Solarbio, Beijing, China) and incubated with the appropriate secondary antibody. The primary antibodies used for Western blot were β-catenin rabbit polyclonal antibody (1:2,000) (ab6302, Abcam, Cambridge, UK), Myc-tag rabbit monoclonal antibody (1:5,000) (AE070, ABclonal), and HA-tag rabbit monoclonal antibody (1:1,000) (C29F4, Cell Signaling). β-actin rabbit monoclonal antibody (1:2,000) (AF5003, Beyotime) was used as a loading control. The secondary antibody was goat anti-rabbit IgG (1:5,000) (A0208, Beyotime). The blot signal was visualized using ECL reagent (Thermo Fisher Scientific, Carlsbad, CA). The protein expression level was quantified by the band density using Quantity One 4.6.3 software (Bio-Rad Laboratories, Hercules, CA) and normalized by β-actin.
3
Streptozotocin-Induced Diabetic Rat Model
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Streptozotocin (STZ), gallic acid, andrographolide, neutral buffered formalin, Harris hematoxylin solution, eosin Y, and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody were purchased from Sigma-Aldrich (Selangor, Malaysia). Sodium azide and all organic solvents were purchased from Merck (Selangor, Malaysia). Rat Insulin ELISA kit was purchased from Mercodia (Sweden). CHOL2 total cholesterol test kit and TRIGL (GPO-PAP) triglyceride test kit were procured from Roche (Germany). RIPA lysis buffer, 2 × protein loading buffer, Tris-buffered saline with 0.1% (v/v) Tween-20, and BCIP/NBT chromogenic substrate kit were purchased from Solarbio (China). PVDF membrane was procured from Bio-Rad (USA). Rabbit anti-rat GLUT4 primary antibody was procured from Cusabio (China).
4
Western Blot Analysis of NCKAP1
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After collecting the transfected cells, they were washed using PBS (Solarbio, Beijing, China). The supernatant was collected after lysis to obtain the total protein. The protein concentration was determined using the BCA protein assay kit (Solarbio, Beijing, China). After separation using 12% SDS-page (Beyotime, Shanghai, China), the cells were transferred to a PVDF membrane (Thermo Fisher, Carlsbad, CA, USA). The membrane was closed for 1 h at room temperature using the Tris-Buffered saline (Solarbio, Beijing, China) containing 5% skim milk powder. Next, the cells were incubated overnight with rabbit anti-NCKAP1 antibody (1:1000, ab126061, Abcam, Shanghai, China) or β-Actin (1:5000, ab179467, Abcam, Shanghai, China). On the following day, after rinsing with TBST, the cells were incubated with goat anti-rabbit IgG H&L (HRP) (1:2000, ab205718, Abcam, Shanghai, China) for 1 h at room temperature, and images were obtained using an infrared laser scanning imaging system (CDYSSEY CLx; General Electric).
5
Western Blot Protein Analysis Protocol
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First, cells were gathered and lysed using radioimmunoprecipitation assay buffer (Beyotime, Jiangsu, China) based on the manufacturer’ protocol. Then the membrane was sealed by 5% skim milk powder for 1 h. Following that, the membrane was incubated with the primary antibodies overnight at 4°C. Then, Tris-buffered saline containing 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to wash the membrane three times for 5 min each. The membrane was probed with the secondary antibody for 1 h at room temperature and washed and added enhanced chemi-luminescence (ProteinTech Group, Inc., Chicago, IL, USA). GAPDH was used as the internal control. The QUANTITY ONE software (version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to scan the gray value.
6
Western Blot Analysis of RUNX-2 Expression
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Following treatment, the protein extracts of the cells were harvested in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration in the cell lysates was quantified using the BCA Protein Assay kit. Proteins (15 µg) were then separated by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes, which were blocked with 2.5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in Tris-buffered saline with 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h. Membranes were then incubated with the following primary antibodies at 4˚C for 15 h: Anti-RUNX-2 (1:200; cat. no. sc-390351) and anti-β-actin (1:1,000; cat. no. sc-8432) (both from Santa Cruz Biotechnology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at 37˚C for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection reagent (Pierce; Thermo Fisher Scientific, Inc.). β-actin in the cell lysates was used as a loading control and data were normalized against the corresponding optical density of β-actin. ImageJ 1.48 software (National Institutes of Health) was used for semi-quantification of bands.
7
Western Blot Analysis of Nanog Protein
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The monoclonal anti-Nanog antibody from medaka was provided by Professor Hongyan Xu [36 (link)]. The total protein of tissues was extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China) and mixed with SDS-PAGE Sample Loading Buffer (Beyotime). After being boiled for 5 min, 10 µL protein buffer was loaded into a lane, electrophoresed through 10% SDS-PAGE gels, and electroblotted onto polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA) by an electroblotter (BioRad, Hercules, CA, USA). The membrane was blocked with 5% BSA (Solarbio) for 1 h. After being washed with TBS (Solarbio), the membrane was incubated with anti-β-Actin antibody (Bioss, Beijing, China) or anti-Nanog antibody (1:1000 dilution in TBS) at 4 °C overnight. Then, the membrane was washed with TBS and incubated with HRP-conjugated goat anti-rabbit IgG (Bioss) (1:2000 dilution in TBS) for 2 h. Finally, protein blots were colored with Chemiluminescent Substrate for Western blotting Kit (Cyanagen, Bologna, Italy) and imaged by an Alliance MINI HD9 system (Uvitec, Cambridge, UK). The β-Actin was used as an internal control.
8
Protein Quantification and Western Blotting
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The total protein content was extracted from midbrain tissue and BV2 cells by using a lysis buffer containing protease inhibitors after washing with PBS three times. The protein concentration was measured with the BCA protein assay kit (Solarbio, pc0020). Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TBS (Solarbio) with Tween 20 (Sigma, P9416) and 5% skim milk for 2 h. Then, the PVDF was incubated with the following primary antibodies overnight at 4°C: TH (1:1,000), IBA-1 (1:800), NLRP3 (1:1,000), ASC (1:400), caspase-1 (1:800), Arg-1 (1:1,000), β-actin (1:2,000). After washing with TBST, the membrane was incubated with horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG (1:2,000) for 1 h at room temperature. At last, the PVDF was treated with enhanced ECL reagent and protein bands were visualized on X-ray film utilizing enhance chemiluminescence system.
9
Visualizing PDCoV Virion Particles
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To image the virion particles of PDCoV, electron microscopy was used, and samples were prepared according to methods described in a previous study (Liu et al., 2019 (link)). Briefly, LLC-PK cells infected with PDCoV were harvested when CPEs were observed in more than 90 % of the cells. The harvested cell cultures were frozen and thawed three times and then centrifuged at 8500×g at 4 °C for 30 min. The cell debris in the supernatant was removed by filtration through a 0.22-mm filter, and then polyethylene glycol 8000 (PEG-8000; Solarbio, China) was incubated overnight at a 10 % final concentration. After that, the suspensions were ultracentrifuged at 12,000×g at 4 °C for 2 h to pellet the viral particles. The purified viral particles were resuspended using Tris buffered saline solution (TBS, Solarbio, China) and then negatively stained with 2% phosphotungstic acid. The image was observed with a transmission electron microscope (JEM-1200EX, Japan).
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