Briefly, proteins were collected from the tendon-bone transition area and articular cartilage using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013C, Beyotime, Shenyang, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF; ST506, Beyotime). A BCA protein assay kit (P0010; Beyotime) was used to quantify the proteins, and 30 μg protein per sample was subjected to polyacrylamide gel electrophoresis. Next, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using wet blotting. The membranes were incubated overnight at 4°C with the following primary antibodies after blocking non-specific binding sites: mouse monoclonal anti-collagen I (1:3,000; 66761-1-Ig, Proteintech), mouse monoclonal anti-collagen II (1:3,000; ab185430, Abcam, United States), and mouse monoclonal GAPDH (1:1,000; 60004-1-Ig; Proteintech). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies at 22 ± 2°C for 2 h. Enhanced chemiluminescence reagent was used to detect immunoreactivity. Finally, Image Pro Plus version 6.0 software (Media Cybernetics) was used to calculate the band intensities.
St506
Manufactured by Beyotime
Sourced in China
The ST506 is a laboratory equipment designed for general laboratory use. It functions as a Digital Multimeter, capable of measuring various electrical parameters such as voltage, current, and resistance.
Automatically generated - may contain errors
Lab products found in correlation
P0013, by Beyotime (23 mentions)
Ipvh00010, by Merck Group (12 mentions)
Ripa lysis buffer, by Beyotime (10 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
P1005, by Beyotime (5 mentions)
A0208, by Beyotime (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Bca kit, by Beyotime (4 mentions)
A0216, by Beyotime (4 mentions)
P1081, by Beyotime (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Wbkls0100, by Merck Group (4 mentions)
P1050, by Beyotime (4 mentions)
Sc 3879, by Santa Cruz Biotechnology (3 mentions)
Sc 33652, by Santa Cruz Biotechnology (3 mentions)
P0015, by Beyotime (3 mentions)
Sa00001 1, by Proteintech (3 mentions)
Iseq00010, by Merck Group (3 mentions)
101 protocols using st506
1
Protein Expression Analysis of Tendon-Bone Transition
2
Quantifying Alzheimer's Biomarkers in PAP Mice
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The cerebral cortex and hippocampus tissues of PAP mice were homogenized in radio immunoprecipitation assay lysis buffer (P0013B, Beyotime, China) containing protease inhibitors (ST506, Beyotime, China), sonicated briefly, lysed on ice for 30 min with gentle agitation, and then centrifuged at 14,800 × g for 20 min at 4°C to remove debris. Aliquots of the lysates were tested for Aβ1-42 monomers and Aβ oligomers, using the Human/Rat Beta Amyloid (42) ELISA kit (290–62601, Wako, Japan) and Mouse Aβ Oligomer ELISA kit (AB6094, Abmart, China), respectively, according to the manufacturer’s protocols. Plasma and brain tissue IL–1β and TNF–α were determined using the Mouse IL-1β (EMC001b, NeoBioscience, China) and TNF-α (EMC102a, NeoBioscience, China) ELISA kits in accordance with the manufacturer’s protocols. In addition, feces of PAP mice were homogenized at 50 mg/mL in sterilized PBS, and large debris were removed by brief pulsed centrifugation. The fecal suspension was centrifuged at 14,800 × g for 15 min at 4°C to remove sediments, and the levels of fecal LCN2 and albumin were assayed using the Mouse LCN-2/neutrophil gelatinase-associated lipocalin (NGAL) Quantikine ELISA kit (MLCN20, R&D Systems, USA) and Mouse Albumin ELISA kit (E13878m, Cusabio, China).
3
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer (Beyotime, P0013B) with protease inhibitors (Roche, 04693132001) and PMSF (Beyotime, ST506). After quantification using a BCA protein assay kit (Beyotime, P0012S), 40 μg of total protein was separated with 7.5% SDS-PAGE under denaturing conditions and was transferred to nitrocellulose membranes (PALL, 66485). The membranes were blocked and then incubated with the primary antibody overnight at 4°C, followed by incubation with anti-rabbit or anti-mouse conjugated antibodies. Antibodies are listed in the supplemental experimental procedures .
4
Lung Tissue Protein Extraction and Western Blot
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Lung tissue homogenate in lysate (P0013B, Beyotime, China) with protease inhibitors (ST506, Beyotime, China), centrifuged for 10 minutes at 4°C, 12000 rpm. The extracted supernatant is boiled and denatured after being treated with a 5 × loading buffer. Protein samples are separated by 10% SDS-PAGE electrophoresis, followed by protein transfer from gel to a PVDF membrane (IPVH00010, Millipore, USA). After 1 hour of blocking with 5% skim milk (Q/NYLB0039S, Yili, China), the membrane is incubated with the primary antibody overnight at 4°C. After three PBST washes of the membrane on the primary antibody, it is incubated for 2 hours at room temperature with the appropriate secondary antibody. Finally, the ECL kit (E002-5, Seven Seas Biology, China) was used to generate protein band pictures on the membrane. A semiquantitative study was conducted using an Image J software.
5
Protein Quantification and Western Blot
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The sample is lysed with 400 µl lysis buffer (P0013, Beyotime Institute of Biotechnology) containing 100 mmol/L PMSF (ST506, Beyotime Institute of Biotechnology) for 30 minutes, then transfer to the EP tube. Centrifuge for 10 min in a high-speed centrifuge at 2000 r/min. The protein concentration was determined according to the BCA kit. The protein was denatured, loaded, and subjected to sodium dodecylbenzene sulfonate gel electrophoresis (SDS-PAGE) for 2 hours, and then transferred to the membrane with a constant current of 300 mA for 80 min. Incubate the primary antibody solution at 4°C overnight; incubate the secondary antibody solution at room temperature for 2 hours. Drop the ECL luminescent liquid on the lm and expose it in the gel imaging system (Chemi DocTM XRS+, Bo Le Biomedical Products (Shanghai) Co., Ltd.). Use "ImageJ" software to analyze the gray value of each band.
6
Western Blot Analysis of Bone-Related Proteins
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, P0013C) containing PMSF (Beyotime, ST506) on ice. Then, 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was used to separate the proteins in the lysates. Proteins were then transferred to PVDF membranes (0.22 μm, Millipore) and blocked with free quick blocking buffer (Epizyme, PS108P) for 15 min at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies and washed thrice with TBST before incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime) at room temperature for 1 h. ECL reagent (Epizyme) was used for the detection of antibody–antigen complexes. Antibodies against Myc (#2272), HA (#3724), Flag (#14793), Runx2 (#12556), TRAF6 (#8028), NFATc1 (#8032), c-FOS (#2250), β-actin (#4970), PI3K (#4257), p-PI3K (#17366), Akt (#4691), p-Akt (#4060), MDM2 (#86934), p53 (#2527), Nedd4 (#5344) were obtained from Cell Signaling Technology (Boston, USA). Antibodies against OSX (#ab209484), OCN (#ab133612) were purchased from Abcam (Cambridge, UK).
7
Protein Extraction and Western Blot Analysis
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Proteins were extracted from rat femur tissues or cells with lysis solution (P0013, Beyotime, China) containing 1% PMSF (ST506, Beyotime). The protein concentration was determined by the BCA kit (P0011, Beyotime). After denaturation and SDS-PAGE electrophoresis, separated proteins were transferred onto PVDF membranes (IPVH00010, Millipore, USA). The membranes were blocked with 5% nonfat milk in TBST for 1 h, and incubated overnight at 4°C with the primary antibodies against OCN (DF12303, 1:500, Affinity, China), collagen-l (AF0134, 1:500, Affinity), RUNX2 (AF5186, 1:1000, Affinity), and β-actin (sc-4778, 1:1000, Santa Cruz, USA). After washing with TBST, the membranes were incubated for 45 min at 37°C with secondary antibodies including HRP-labeled goat anti-rabbit IgG (A0218, 1:5000, Beyotime) and HRP-labeled goat anti-mouse IgG (A0216, 1:5000, Beyotime). The immunoreactive protein was visualized by a WD-9413B gel imaging system (Beijing Liuyi, China) with ECL kit (P0018, Beyotime).
8
Proteomic Analysis of Coronary Arteries
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Coronary artery tissues were frozen in liquid nitrogen. Total protein was extracted from Coronary artery using RIPA lysis buffer (P0013B, Beyotime) with PMSF (ST506, Beyotime), resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting. CTSB/C/D/Z proteins were detected using monoclonal antibody, then they were subjected to horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG polymer (1:5000). After the addition of developer and post-exposure, grayscale values were measured using ImageJ analysis software (National Institutes of Health, USA) and internal controls were used with β-actin.
9
Quantitative Protein Analysis via Western Blot
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Total proteins from the cultured cells were extracted using RIPA Lysis Buffer (P0013B, Beyotime, China,) containing PMSF (ST506, Beyotime, China,) and protease inhibitors. BCA assay kit (E-BC-K318-M, Elabscience) was used to determine the protein concentration. Proteins were subjected to SDS-PAGE and transferred onto PVDF (Millipore, USA). Following, antigen blocking with 5% BSA, the blots were incubated with primary antibodies overnight at 4 ℃, followed by secondary antibodies or HRP-labelled secondary antibodies at room temperature for 1 h. The final detection of immunoreactive bands was developed using an enhanced chemiluminescent Western blot system (ECL) (BL520A, biosharp China) with exposure to a chemiluminescence imaging system (UVP). The immunoblotting signal intensity was measured using ImageJ 64 software.
10
Western Blot Analysis of Protein Extracts
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Total protein was extracted with cell lysis buffer (P0013, Beyotime, Shanghai, China) containing PMSF (1:100, ST506, Beyotime, Shanghai, China) and a protease inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China). A BCA protein assay kit (CW0014S, CWBiotech, Beijing, China) was used to measure the protein concentration. The protein lysates were electrophoretically separated on sodium dodecyl sulfate–polyacrylamide gels and electrically transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, Ireland). The membranes were blocked in 5% skimmed milk for 1 h and incubated with the primary antibodies (Additional file 7 : Table 4) for one night at 4 °C. Then, the membranes were incubated with secondary antibodies (Additional file 7 : Table 4) at room temperature for 1 h. Proteins were visualized using the Tanon 5200 chemiluminescence imaging system following incubation with BeyoECL Plus (P0018S, Beyotime, Shanghai, China).
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