The site was made moisture less by syringe (air) to removing the contamination of saliva, after insulating the implant with gauze. Then, PICF was possessed by putting 2 mm strips of standard paper into the pocket/sulcus for 30 s. The visually contaminated strips by blood were thrown away. PICF strips were inserted with 250 ml phosphate‐buffered saline (PBS) and a cocktail of protease inhibitors (Beyotime, Shanghai, China) into microfuge tubes. Then, the samples were stored at −70°C for further analysis.
Cocktail of protease inhibitors
Manufactured by Beyotime
Sourced in China
The Cocktail of Protease Inhibitors is a mixture of compounds designed to inhibit the activity of proteases, which are enzymes responsible for the breakdown of proteins. The product is intended for use in research applications where the preservation of protein integrity is crucial.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti gapdh antibody, by Vazyme (1 mentions)
Luminescent imaging system, by Bio-Rad (1 mentions)
Ecl chemiluminescence kit, by Sartorius (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
β tubulin, by Solarbio (1 mentions)
Amersham imager 6000, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
5 protocols using cocktail of protease inhibitors
1
Saliva-Free Implant Pocket Sampling
2
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from fresh tissues and cell lines with RIPA lysis buffer containing a cocktail of protease inhibitors (Beyotime Institute of Biotechnology). The protein samples were then separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes (Millipore). After blocking with 5% nonfat milk dilution with TBST for 1 h at room temperature, the membrane was incubated with primary antibodies against INPP4B, p-SGK3T320, SGK3, p-AKTT308, and AKT at 4°C overnight. After washing with TBST, the membranes were incubated with the secondary antibody (Cell Signaling Technology) at a dilution of 1:3000 at room temperature for 60 min. The membranes were then washed with TBST, and the bound antibodies were detected with an enhanced chemiluminescence system. Anti-GAPDH antibody (1:3000; Vazyme) was used as a loading control.
3
Western Blot Analysis of UMSC Proteins
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Protein samples were extracted from UMSCs or exosomes using RIPA buffer (Beyotime Biotechnology) containing a cocktail of protease inhibitors (Beyotime Biotechnology). Equal amounts of protein were separated by SDS‐polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated with primary antibodies followed by a horseradish peroxidase‐conjugated secondary antibody. The primary antibodies used were B2M (1:5000; Abcam Inc) and Bim (1:1000; Cell Signaling Technology Inc). The proteins were visualized using an ECL chemiluminescence kit (Biological Industries), and the luminescence was detected using a BioRad luminescent imaging system.
4
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells by using RIPA Lysis Buffer (Beyotime, China) supplemented with a cocktail of protease inhibitors (Beyotime, China) after rinsing cells three times with cold PBS. The lysates were then subjected to centrifugation at 12,000×g at 4 °C for 5 min. Next, the isolated protein samples were mixed with sample buffer and boiled for 5 min at 100 °C. The total protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Equivalent amounts of protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Sigma, USA). After blocking with 5% skimmed milk, the membranes were incubated with the following antibodies: MMP17 (1:1000, Zenbio), SH3GL2 (1:1000, Zenbio), and β-Tubulin (1:1000, Solarbio). The membranes were then incubated with appropriately HRP-conjugated secondary antibodies (1:5000) and extensively rinsied with TBST. Immunoreactive bands were detected using a chemiluminescence kit (ECL Substrate kit; Abclonal, China), and the protein bands were captured using the Amersham Imager 6000 (GE Healthcare).
5
RIG-I and DDX21 Interaction Assay
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HEK-293T cells cultured in a 60-mm plate were transfected with 3 µg of pCAGGS-Flag-RIG-I, pCAGGS-Flag-DDX21, or empty vector for 24 h. The cells were collected and lysed in 400 µl lysis buffer supplemented with a cocktail of protease inhibitors (Beyotime). The cellular lysates were mixed with the prepared poly (I: C)-coated agarose bead suspension and incubated at 4°C for 4 h. The beads were centrifuged and washed three times with 1 mL of lysis buffer, followed by western blot assay using mouse anti-Flag antibody as the primary antibody.
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