Mice tissues were isolated and homogenized in RIPA buffer (Cell Signaling) containing serine protease inhibitor (Sigma). Homogenates were centrifuged at 1500 g for 5 min and the supernatant was collected and stored at −80 °C. Protein concentration was measured using the Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Hercules, CA). Proteins were resolved on 12% BT SDS-PAGE gels (Invitrogen) and electrotransferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-AQP4 or rabbit anti-CD59 antibody (Santa Cruz Biotechnology) or anti-β-actin antibody (GE Healthcare, Piscataway, NJ) followed by horseradish peroxidase-linked anti-rabbit IgG or anti-mouse IgG (1:10.000, GE Healthcare), and visualized by enhanced chemiluminescence (GE Healthcare).
Serine protease inhibitor
Manufactured by Merck Group
Sourced in United States
Serine protease inhibitor is a type of lab equipment used to inhibit the activity of serine proteases, a class of enzymes that hydrolyze peptide bonds in proteins. It functions by binding to and blocking the active site of serine proteases, preventing them from carrying out their catalytic activities. This equipment is commonly employed in research and diagnostic applications that involve the study of serine protease-mediated biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Protein assay kit 2, by Bio-Rad (1 mentions)
Genegnome xrq system, by Syngene (1 mentions)
Anti psapk jnk, by Abcam (1 mentions)
Anti p38, by Abcam (1 mentions)
Anti p p38, by Abcam (1 mentions)
Anti gapdh primary antibody, by Abcam (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti ikkβ, by Abcam (1 mentions)
Loading buffer, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by CWBIO (1 mentions)
Anti p ikkα β, by Abcam (1 mentions)
Anti ikkα, by Abcam (1 mentions)
Anti sapk jnk, by Abcam (1 mentions)
Anti p p65, by Abcam (1 mentions)
Anti p65, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
5 protocols using serine protease inhibitor
1
Quantitative Analysis of AQP4 and CD59
2
Protein Extraction and Western Blot Analysis
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After removing the supernatant, the cells were washed twice with PBS at 4°C. RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor (Sigma–Aldrich, USA) and 1% serine protease inhibitor (Sigma–Aldrich, USA) was added for 30 min to lyse cells and extract proteins from each sample. The concentrations of total proteins were detected by a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (ThermoFisher, USA) at a ratio of 4:1 and boiled at 99°C for 10 min. Samples were loaded and run on SDS–PAGE gels (CWBIO, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 2% skim milk (BD, USA) for 1 h at room temperature and then incubated with anti-IKKα, anti-IKKβ, anti-pIKKα/β, anti-p65, anti-pp65, anti-p38, anti-pp38, anti-SAPK/JNK, anti-pSAPK/JNK, and anti-GAPDH primary antibodies (Abcam, UK) overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit or goat anti-mouse HRP (Abcam, UK) for 1 hour. Membranes were washed and exposed to chemiluminescent HRP substrate (Millipore, USA). Images were obtained using the GeneGnome XRQ system (Syngene, USA) and analyzed using ImageJ software.
3
Western Blot Analysis of PDCD4 Protein
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Transfected cells were harvested and lysed with CellLytic M cell lysis reagent (Sigma-Aldrich) supplemented with 0.1% serine protease inhibitor (Sigma-Aldrich). The protein concentration was determined using the Bradford assay (Bio-Rad) and the Qubit 2.0 fluorometer (Life Technologies), with bovine serum albumin (BSA) acting as a standard. Twenty micrograms of protein was loaded onto a precast NuPAGE (4 to 12%) Bis-Tris gel (Invitrogen) in NuPAGE morpholineethanesulfonic acid (MES)-SDS running buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA). After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Pierce) using NuPAGE transfer buffer (25 mM Bicine, 25 mM Bis-Tris, 1 mM EDTA). The membrane was incubated with PDCD4 (1:5,000) or tubulin (1:1,000), followed by conjugation with a secondary antibody containing horseradish peroxidase (HRP). Chemiluminescence with the ECL plus reagent (GE Healthcare Life Sciences) was used to visualize the specific protein bands.
4
Western Blot Analysis of Liver Proteins
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Cells were trypsinized and collected. Protein was extracted using RIPA buffer (Cell Signaling) with serine protease inhibitor (EMD Millipore) and phenylmethanesulfonylfluoride (PMSF; Cell Signaling). Homogenates were centrifuged, supernatants were collected and stored at −80°C. Proteins were resolved on a 10% Mini-PROTEAN TGX precast SDS-PAGE gel (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 10% non-fat dry milk in PBS for 2 hours, followed with overnight incubation at 4°C with the following primary antibodies: anti-human albumin (Sigma-Aldrich), anti-human AFP (Dako), or anti-human GAPDH (Cell Signaling). After 1 hour of incubation with HRP-conjugated secondary antibody (Cell Signaling), the signal was detected using ECL reagents (Thermo Pierce). Porcine liver tissues were used as a positive control.
5
Infant Appetite Hormone Profiles
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At age 3 months, 297 blood samples were collected by toe prick after the infants had fasted for a minimum of 2 h. For 184 of these infants, we also collected a blood sample at age 6 months. This number was less than at 3 months because infants were either too distressed to allow a blood collection or had not yet reached age 6 months. Blood samples were collected in EDTA tubes and DPP4 (dipeptidyl peptidase-4) inhibitor, Serine Protease inhibitor and Protease inhibitor (all Merck Chemicals Netherlands—Merck KGaA) were added for stabilizing the appetite-regulating hormones. Blood was centrifuged at 4 °C to prepare plasma, which was quickly frozen and stored at − 80 °C until analyses. Ghrelin (acylated), PYY and leptin levels in plasma were determined by the MILLIPLEX MAP Human Metabolic Hormone Magnetic Bead Panel, catalog number HMHEMAG-34 K (Millipore Corporation, Billerica, MA) using the commercial protocol provided by the supplier. The intra-assay CV was 10%, and the inter-assay CV was 15%. Fasting time was calculated as time of blood collection minus time of last feeding.
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