Western blot analysis was performed as previously described (Liu et al., 2017 (link)). Briefly, after washing with ice-cold PBS, RIPA buffer (Beyotime Institute of Biotechnology) with phosphatase as well as protease inhibitors (cocktail tablet; Roche Applied Science) was utilized to lyse A7r5 and HAMSC cells. After centrifuging at 12,000 ×g at 4°C for 15 min, supernatants were collected, followed by the determination of protein concentration of each sample using BCA protein assay kit (Thermo) according to the manufacturer’s instruction. Protein samples (10 μg) were subjected to SDS-PAGE, and subsequently transferred onto PVDF membrane (Millipore, Bedford, MA, United States). After blocking in 5% non-fat milk, the membranes were incubated with indicated primary antibodies overnight (p-AMPKα, p-mTOR, AMPKα, mTOR, p-p70SK6, p-4EBP1, CyclinD1, CyclinD3, CyclinA2, CDK2, CDK4, CDK6, Bax, Bcl-xl, cleaved-caspase3 and Pro-caspase3, all at a dilution of 1:1000). And GAPDH was used as the internal control (dilution 1:5000). Finally, ImageJ software was used to quantify the band intensity, followed by normalization with the intensity of internal control.
Protease inhibitors cocktail tablet
Manufactured by Roche
Sourced in Switzerland, United States
Protease inhibitors cocktail tablets are a product designed for use in laboratory settings. They function as a mixture of compounds that inhibit the activity of proteases, which are enzymes that cleave proteins. The tablets are intended to be used as a research tool to study protein interactions and stability.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polylysine, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
D trehalose dihydrate, by Merck Group (1 mentions)
Anti flag m2 affinity beads, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Anti sqstm1 p62, by Abcam (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Peasy basic seamless cloning and assembly kit, by Transgene (1 mentions)
Anti flag, by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
16 protocols using protease inhibitors cocktail tablet
1
Western Blot Analysis of Cellular Signaling
2
Protein Purification and Immunoblotting Protocol
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The following reagents were used: 4,6-diamidino-2-phenylindole (DAPI) (D9542; Sigma-Aldrich), BCA protein assay kit (23250; Thermo Fisher Scientific), BSA (New England BioLabs), D-(+)-trehalose dihydrate (T9531; Sigma-Aldrich), ECL detection reagents (32106; Thermo Fisher Scientific), EtBr (E1385; Sigma-Aldrich), GSH (G2451; Sigma-Aldrich), imidazole (I5513; Sigma-Aldrich), immunoglobin G (IgG) (AC011, mouse; Abclonal), polyethylenimine (PEI) (Polysciences), paraformaldehyde (PFA) (158127; Sigma-Aldrich), poly-lysine (P4707; Sigma-Aldrich), protein G-Sepharose CL-4B beads (17–0618-01; GE Healthcare), protease inhibitors cocktail tablet (4693132001; Roche), restriction enzymes including EcoRI and BamHI (New England BioLabs), and RIPA buffer (#9806; Cell Signaling Technology). Antibodies including anti-Flag (F3165, mouse monoclonal; Sigma-Aldrich), anti-HA (C29F4, rabbit monoclonal; Cell Signaling Technology), anti-Flag M2 affinity beads (A2220; Sigma-Aldrich), and anti-SQSTM1/p62 (ab56416, mouse monoclonal; Abcam; #7695, rabbit polyclonal; Cell Signaling Technology, respectively) were used in this study. pEASY-Basic Seamless Cloning and Assembly Kit (CU201; TransGen Biotech), Trans2K Plus II DNA Markers (BM121; TransGen Biotech) and Page Ruler Prestained Protein Ladder (26616; Thermo Fisher Scientific) were also used in this study.
3
Preparation of Mouse Brain Homogenates
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The procedure used to prepare mouse brain homogenate is the same as described in our earlier study25 . The mouse brain tissues were snapped frozen in liquid nitrogen when harvested and the wet weight of the tissues (in mg) was determined using an electronic balance. Twenty percent (w/v) brain homogenates were prepared with ice-cold 1 × RIPA lysis buffer (Cell Signaling Technology) containing detergents such as 1% Nonidet P40 and 1% sodium deoxycholate together with the protease inhibitors cocktail tablet (Roche). This lysis buffer also contains sodium orthovanadate, pyrophosphate and glycerophosphate, which can act as phosphatase inhibitors.
Lysates were homogenized using a hand held motorized pestle (Sigma-Aldrich, St. Louis, USA) for 30 seconds on ice. Tissue lysates were subsequently centrifuged at 30,000 g for 30 minutes under 4°C. The soluble portion of the lysates was collected for analysis.
Lysates were homogenized using a hand held motorized pestle (Sigma-Aldrich, St. Louis, USA) for 30 seconds on ice. Tissue lysates were subsequently centrifuged at 30,000 g for 30 minutes under 4°C. The soluble portion of the lysates was collected for analysis.
4
Antimicrobial Evaluation of Pancreatic and Duodenal Secretions
5
Red Blood Cell Membrane Protein Analysis
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RBC membranes were obtained by lysing one volume of packed RBCs in 10 volumes of ice-cold lysis buffer (5 mM Na2HPO4, pH 8.0 with protease inhibitors (cocktail tablet; Roche, Basel, Switzerland), 3 mM benzamidine and 1 mM Na3VO4) for 10 min on ice. The membrane fraction was washed repeatedly by centrifugation at 21,000xg for 10 min to remove free hemoglobin and solubilized 1:1 with Laemmli buffer (Bio-Rad Laboratories, Hercules CA, USA) supplemented with 100 mM β-mercaptoethanol for SDS-PAGE. SDS-PAGE was carried out according to Laemmli [11 (link)] on 10% polyacrylamide gels. Proteins were transferred to PVDF membranes using the iBlot system (Invitrogen, Carlsbad CA, USA). These membranes were blocked with Odyssey Blocking Buffer (LI-COR, Lincoln NE, USA) and probed with 1:2000 mouse anti-band 3 N-terminal domain (BIII-136, Sigma-Aldrich, St. Louis MO, USA), 1:5000 mouse anti-β-spectrin (Acris, Herford, Germany), or 1:10000 anti-β-actin (Sigma-Aldrich). As secondary antibodies 1:10000 goat anti-rabbit IgG-Alexa Fluor 680 (Invitrogen, Carlsbad CA, USA), and/or goat anti-mouse IgG-IRDye 800 (LI-COR, Lincoln NE, USA) were used. The blots were scanned with the Odyssey Infrared Imaging System (LI-COR, Lincoln NE, USA) and the images were analyzed using the Odyssey Software version 2.1.
6
Protein Extraction from Fish Samples
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Protein preparation. Five aquacultured sea bream and five L3 A. simplex larvae-infected horse mackerel, previously stored at −20 °C for 24 h, were utilized for the study. Protein preparation was conducted mainly following the procedure used for L3 A. simplex larvae. Of each fish, 2 g of edible part was weighed and homogenized with 7 mL of RIPA buffer (Sigma-Aldrich, Milan, Italy), containing protease inhibitors cocktail tablets (Roche Applied Science, Milan, Italy) and anti-phosphatases (sodium orthovanadate 2 mM; Sigma-Aldrich, Milan, Italy). The homogenate was then centrifuged at 16,000× g for 30 min and separately aliquoted and stored at −20 °C until the use.
7
Immunoprecipitation of Target Proteins
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Beads used in the immunoprecipitation were washed and equilibrated in 0.1% NP-40 lysis buffer. Briefly, cell lysates were prepared using immunoprecipitation assay buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% NP-40, pH=7.2–7.4, protease inhibitors cocktail tablets (Roche)) for 30 min at 4℃, then incubated with anti-flag beads (Sigma) to enrich the target protein overnight at 4℃. For immunoprecipitaion of TRIP12-myc protein, the cell lysates were first incubated with anti-myc antibody overnight at 4℃, followed by 4 hours incubation with protein A/G plus-agarose beads. The beads were then washed vigorously with immunoprecipitation lysis buffer for 6–8 times. The protein was eluted by boiling the beads with SDS loading buffer for 5 min, and then subjected to western blot analysis.
8
Protein Extraction and Western Blot Analysis
9
Co-immunoprecipitation of Cellular Proteins
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Co-immunoprecipitation experiments were performed using lysates from Caco-2bbe cells or mouse intestinal mucosa prepared using lysis buffer (Cell Signaling, Danvers, MA) containing 150 mmol/L NaCl, 1 mmol/L β-glycerophosphate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L Na2 EDTA, 1 mmol/L EGTA, 1 mmol/L Na3VO4, 1 μg/mL leupeptin, 1% Triton X-100, and protease inhibitors cocktail tablets (Roche, Indianapolis, IN). Lysate (500 μg) was pre-cleared by incubation with 30 μL of protein A-Sepharose beads for 1 hour, and the supernatant was then incubated overnight with a primary antibody (anti-HA, anti-VSVG, or anti-NHE3 antibody). Immunocomplex was purified by incubating with 50 μL of protein A-Sepharose beads for 1 hour, followed by 3 washes in lysis buffer and 2 washes in phosphate-buffered saline. All of the above steps were performed at 4°C or on ice. Beads eluted with 2.5× Laemmli buffer. Cell lysates and eluted samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western immunoblotting and quantification were performed as described previously.17 (link),25 (link)
10
Tissue Homogenization and Protein Extraction
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Mice were euthanasia with 5% isoflurane (RWD, Shenzhen) and perfused intracardially with 30 ml cold PBS. Then, the brain, liver, heart, and spleen were homogenized in RIPA lysis buffer (Beyotime Biotechnology, P0013B) or NP-40 lysis buffer (Beyotime Biotechnology, P0013F). For proteome and acetylome analysis, mice brains were homogenized in urea lysis buffer (8 M sequencing grade urea, 100 mM Tris-HCl PH 8.0). Protease inhibitors cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) were added to all three protein lysis buffers. Centrifuge the lysis at 10,000 g for 15 min at 4°C, and the supernatant was collected and stored at -80°C. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Pierce BCA Protein Assay Kit, Cat#23225).
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