The treated or untreated cells were collected, and the total protein was extracted by RIPA reagent (P0013B, Beyotime) with 1% protease inhibitors (P1030, Beyotime). After that, the concentration of the protein was determined by BCA kit (P0012, Beyotime). Then, cellular protein of 30 μg was separated by SDS-PAGE gel (P0012A, Beyotime). After electrophoresis for 90 min, the proteins were transferred on PVDF membranes (ISEQ00010/IPVH00010, MILLIPORE, MA, USA). Then, the membranes were blocked by 5% skimmed milk (D8340, Solarbio) for 1 h at room temperature, followed by incubation in primary antibodies (RUNX2, ab23981, 1：1,000, 60 kDa, Abcam, Cambridge, MA, USA; OCN, ab93876, 1：500, 11 kDa, Abcam; BCL2, ab182858, 1：2,000, 26 kDa, Abcam; GAPDH, ab181602, 1：10,000, 36 kDa, Abcam) at 4℃ overnight. Next day, the primary antibodies were recycled, and TBST (ST-673, Beyotime) was used to wash the membranes for 4 times (5 min for each time). Afterwards, secondary antibody (Goat Anti-Rabbit IgG H&L (HRP): ab6721, 1：10,000, Abcam) was incubated the membranes for 1 h (room temperature), followed by washing with TBST for 6 times (5 min for each time). Finally, ECL solution (WBKLS0500, MILLIPORE, Billerica, MA, USA) was dripped onto the membrane, and a specific imaging system (Bio-Rad, CA, USA) was used to visualize the band.
St673
Manufactured by Beyotime
Sourced in China
The ST673 is a laboratory equipment designed for precision measurement and analysis. It is a compact and versatile instrument capable of performing various tasks in a laboratory setting. The core function of the ST673 is to provide accurate and reliable data collection and processing. Further details on its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (1 mentions)
Ripa reagent, by Beyotime (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Iseq00010 ipvh00010, by Merck Group (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
D8340, by Solarbio (1 mentions)
Goat anti rabbit igg h l hrp ab6721, by Abcam (1 mentions)
Ecl reagent, by Vazyme (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
G9023, by Merck Group (1 mentions)
C1002, by Beyotime (1 mentions)
C1090, by Beyotime (1 mentions)
5 protocols using st673
1
Western Blot Analysis of Protein Expression
2
Temporal Bone Protein Extraction and Western Blot
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Mice were sacrificed by cervical dislocation, and the temporal bones were rapidly removed and placed in eppendorf tubes containing a protease inhibitor cocktail (Sigma, 0 469 313 2001) and medium‐strength RIPA lysis buffer (Beyotime, P0013k). Two pre‐cooled magnetic beads were added to the tubes, and the eppendorf tubes were placed in a high‐throughput tissue grinder (Chengk Instruments, Grinder‐48) and ground three times for 1 min with a 1 min interval between each grinding. The tubes were then centrifuged at 14 000× g at 4 °C for 10 min. The supernatant was then mixed with an equal volume of SDS and then boiled in water for 10 min for western blot experiments or stored at −20 °C. Protein samples were soaked in boiling water for 10 min before performing western blotting, and each sample was separated using SDS‐PAGE and then transferred to a PVDF membrane (Millipore, IPVH00010). The membranes were blocked with 5% skim milk in TBST (Beyotime, ST673) for 1 h at room temperature and then incubated with the primary antibody in TBST overnight at 4 °C. The next day, the membrane was washed with PBST three times for 10 min each and then incubated with secondary antibody for 1 h at room temperature and then imaged with ECL reagent (Vazyme, E412‐01).
3
Western Blot Analysis of Protein Samples
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The cells were washed with ice-cold PBS twice and lysed in ice-cold RIPA buffer (Beyotime, P0013B) containing 1% PMSF (Beyotime, ST506) and 1% protease inhibitor cocktail (MCE, HY-K0010) for 15 minutes. After centrifugation (1.3 × 104 rpm, 30 minutes) at 4°C, total protein concentrations of the supernatant were quantified by BCA protein assay kit (Beyotime, P0010). The standard protein samples (40 μg) were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST (Beyotime, ST673) and incubated with secondary antibody for 2 hours at room temperature. After being washed with TBST three times, the protein blots were visualized by enhanced chemiluminescence and autoradiography.
4
Immunofluorescence Staining of PD-L1 in Tissue
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For IF, place paraffin slices in 75°C for half an hour to prevent slices from falling off, dewaxing and hydration. Then, tissue sections were placed vertically in Tris‐EDTA antigen repair solution (ST725, Beyotime, China) and heated for 10 min at 65°C to repair the antigens, and removed to room temperature. After that, blocked it with 10% goat serum (G9023, Sigma–Aldrich, USA) for 1 h at room temperature, and then incubated with DAPI/PI/anti‐PD‐L1 antibodies (DAPI, 5 mg/ml, C1002, Beyotime, China; PI, 5 mg/ml, P0135, Beyotime, China; Anti‐PD‐L1, 1:250, 66248‐1‐Ig, Proteintech, China) overnight at 4°C. After washing with 1× TBST (ST673, Beyotime, China) for 5 min, the sections were incubated with the appropriate secondary antibody (488‐conjugated antibody, 1:500, 115‐545‐146; 680‐conjugated antibody, 1:500, 115‐625‐146, Jackson ImmunoResearch Laboratories, USA) for 30 min at room temperature. For the negative control, sections were incubated with the secondary antibody only. Further detection was done by following the manufacture protocol. Photographs were taken under the same conditions with a fluorescence microscope (Leica, Germany). The experiment was repeated three times.
5
TUNEL Assay for Apoptosis Detection
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Cells were seeded into coverslips and exposed to various treatments. Briefly, cells were fixed with 4% paraformaldehyde, washed with phosphate buffered saline (PBS) (C0221A, Beyotime Biotechnology) twice for 3 min, and then treated with 0.3% Triton X-100 for 10 min. Aliquots of 50 µL TUNEL solution (C1090, Beyotime Biotechnology) were placed in coverslips and cells were cultured for 1 h. Coverslips were washed with tris buffered saline tween (TBST) (ST673, Beyotime Biotechnology) three times. Cells without any substance acted as control. Photographs were taken by microscopy (Olympus BX51). The results of TUNEL were obtained from more than three replicate experiments performed by two operators under the same laboratory conditions. The same reagents and instrument systems were used for all experiments. All methods have mentioned the control in each experiment.
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