For lysing cells and to extract protein RIPA buffer was used, adding a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). For the SDS-PAGE assay, a total of 20 µg/sample was loaded in the acrylamide gel. Proteins were transferred from the gel to 0.2 µm pore-size nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were then blocked for 1 h using 5% BSA (PanReac AppliChem, ITW Reagents) in 1X TBS-T (0.1% Tween20, Bio-Rad). After membrane blocking, the following antibodies were used for blotting proteins: anti-HMGA1 (ab129153; Abcam), anti-PARP1 (51-66396R; BD Bioscience, Franklin Lakes, NJ, USA), anti-cleaved Caspase 3 (#9661; Cell signaling, Danvers, MA, USA), anti-phospho-S6 (#4858; Cell signaling), anti-S6 (#2217; Cell signaling) and anti-α-tubulin (Sigma-Aldrich, T9026). Membranes were consequently incubated with Rabbit Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich) or Goat Anti-Rabbit IgG H&L peroxidase-conjugated antibody (Abcam) secondary antibodies. For chemiluminescent detection, the HRP substrate was used. Image acquiring was performed using the ChemiDoc Imaging System (Bio-Rad). ImageJ was used to quantify protein expression levels.
Cocktail of protease and phosphatase inhibitors
Manufactured by Merck Group
Sourced in United States
The Cocktail of protease and phosphatase inhibitors is a laboratory product designed to prevent the degradation of proteins during sample preparation and analysis. It contains a combination of compounds that inhibit the activity of proteases and phosphatases, which are enzymes that can break down or modify proteins. This product helps to preserve the integrity of proteins in biological samples, allowing for more accurate analysis and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (3 mentions)
M per, by Thermo Fisher Scientific (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse igg peroxidase antibody, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
0.2 m pore size nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti parp1, by BD (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti hmga1, by Abcam (1 mentions)
Anti phospho s6, by Cell Signaling Technology (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg h l peroxidase conjugated antibody, by Abcam (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Odyssey system, by LI COR (1 mentions)
19 protocols using cocktail of protease and phosphatase inhibitors
1
Cell Lysis and Protein Extraction Protocol
2
Western Blot Analysis of Phosphorylated STAT3/5
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Immediately after collection, the hypothalamus and mammary glands were homogenized in RIPA buffer (Sigma) containing a cocktail of protease and phosphatase inhibitors (1:100, Sigma) and centrifuged (14000 RPM, 4 °C for 20 minutes), and the supernatants were retained. After determining the total protein concentration (Pierce BCA Protein Assay, Thermo Scientific), 50 μg of total protein was loaded on a 10% SDS-PAGE gel, and the separated proteins were then transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% bovine serum albumin and incubated overnight at 4 °C using commercially available primary antibodies (1:1000) to identify pSTAT3Tyr705 (Cell Signaling), pSTAT5Tyr694 (Cell Signaling), GAPDH (Santa Cruz) or α-tubulin (Cell Signaling). Next, we incubated the membranes for 45 min in 1:10,000 secondary antibody (IRDye 800CW, Li-COR). Proteins were detected by fluorescence and analyzed using the Li-COR Odyssey system (Li-COR), and protein amounts were normalized to GAPDH or α-tubulin amounts.
3
Quantitative Analysis of Nucleolar Morphology
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We performed immunofluorescence staining of nucleophosmin/B23 (same as immunofluorescence protocol above) in Nthy-SEC23B-WT and Nthy-SEC23B-V594G cells. Nucleophosmin/B23 marks the granular component (GC) of nucleoli. DAPI staining was used to demarcate nuclei. Slides were visualized and images obtained using a Leica DMI3000B manual inverted microscope (Leica, Buffalo Grove, IL, USA). We counted on average approximately 250 cells per genotype, and counting was done on two independent biological replicates.
For quantification of nucleolar mass, we performed Western blot analysis. We extracted protein using the Mammalian Protein Extraction Reagent M-PER (Thermo Scientific Pierce, Rockford, IL, USA) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and quantified through the BCA protein assay (Thermo Scientific Pierce). Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. We probed for anti-UBF mouse monoclonal (Santa Cruz sc-13125) at 1:500, anti-Nucleophosmin/B23 (Abcam ab10530) mouse monoclonal at 1:1000, anti-Fibrillarin rabbit monoclonal (Cell Signaling # 2639) at 1:1000 and anti-GAPDH rabbit monoclonal (Cell Signaling #2118) at 1:20 000 dilutions. Blots were scanned digitally and quantified using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).
For quantification of nucleolar mass, we performed Western blot analysis. We extracted protein using the Mammalian Protein Extraction Reagent M-PER (Thermo Scientific Pierce, Rockford, IL, USA) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and quantified through the BCA protein assay (Thermo Scientific Pierce). Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. We probed for anti-UBF mouse monoclonal (Santa Cruz sc-13125) at 1:500, anti-Nucleophosmin/B23 (Abcam ab10530) mouse monoclonal at 1:1000, anti-Fibrillarin rabbit monoclonal (Cell Signaling # 2639) at 1:1000 and anti-GAPDH rabbit monoclonal (Cell Signaling #2118) at 1:20 000 dilutions. Blots were scanned digitally and quantified using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).
4
GFP, SEC23B, and UBF Immunoprecipitation
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Cells were pelleted and lysed with M-PER (Thermo Scientific Pierce) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Protein lysates were collected by centrifugation at 13 000 rpm for 10 min at 4°C and pre-cleared by incubation with Thermo Protein A/G Dynabeads for 3 h at 4°C on a rotator. Pre-cleared protein lysates were quantified with the BCA Protein Assay Kit (Thermo Scientific Pierce), and 1 mg/ml lysates were prepared. We used anti-GFP (Abcam ab290), anti-SEC23B (Abcam ab151258) and anti-UBF (Santa Cruz sc-13125) antibodies for pull-down and immunoblotting at the recommended dilutions. Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were scanned digitally and quantified using the Odyssey Infrared Imaging System (Li-Cor Biosciences).
5
Protein Expression Analysis in Adipose Tissue
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Cells were lysed in RIPA buffer (Sigma) containing cocktail of protease and phosphatase inhibitors (Sigma). Equal amounts of solubilized proteins were loaded on 4–20% gradient SDS-PAGE gels (Bio-Rad), blotted onto nitrocellulose membranes and incubated with the following primary antibodies: HSL (#4107), ATGL (#2138) and GAPDH (#2118) were purchased from CST Ozyme; total PDHE1α (#Ab110334), phospho-Ser293 PDHE1α (#Ab177461), UCP1 (#Ab10983) and GLUT1 (#Ab40084) from Abcam; PLIN5 (#GP31) and PLIN 1 (#GP29) from PROGEN; PDK4 (#H00005166-A02) from Abnova and PCK1 (#AP8093b) from Abgent. Anti-rabbit or anti-mouse IgG coupled to horseradish peroxidase were used as secondary antibodies. Immunoreactive proteins were determined by chemiluminescence (Clarity, Bio-Rad) with a ChemiDoc MP System (Bio-Rad) and quantification was performed using Image Lab software (Bio-Rad).
6
Western Blot Analysis of Protein Expression
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Cells were harvested in ice-cold RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Lysis was performed on ice for 20 min, and the total lysates were centrifuged at 14,000× g for 10 min (4 °C). A Bio-Rad protein assay was used for protein quantification. Cell lysates (≥30 μg) were resolved by SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat dry skim milk, the membranes were immunoblotted for the desired proteins. GAPDH served as a protein loading control. The immunoblots developed on X-ray film were visualized by an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific), and the intensity of protein bands was quantified using Image J software (NIH, Bethesda, MD, USA).
7
Protein Expression Analysis by Western Blot
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Proteins were extracted with a lysis buffer containing 0.1% Triton X-100 and a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich), and their concentration was measured by Bradford assay (Bio-Rad). The proteins were separated on (8%–12%) SDS-PAGE and transferred onto polyvinylidene fluoride membrane [13 (link)]. Membranes were probed with primary antibodies against transforming growth factor-β (TGF-β), TGF-β receptor (TGF-βR), SMAD3, tumor necrosis factor α (TNFα), interleukin 6 (IL-6), Nox2, Nox4, E-selectin (Abcam), NF-κB, interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) (Santa Cruz Biotechnology), eNOS (Thermo Fisher Scientific), and phospho-SMAD3Ser423/425 (Cell Signaling Technology, Danvers, MA, USA). Loading conditions were determined with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were employed to detect primary antibodies (Santa Cruz Biotechnology). Antibody binding was visualized by chemiluminescence, and images were collected and analyzed using a ChemiDoc-It Imager (Ultra-Violet Products, Cambridge, UK).
8
Cell Lysis and Immunoprecipitation Protocol
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SK CO-15 or HEK 293T cells were harvested after 48 h of transfection in Triton lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Lysates were incubated on ice for 20 min before centrifugation at 16,000 × g for 20 min. Collected supernatants were precleared with 50 μl of 50% protein A or G agarose beads for 1 h followed by incubation with rotation overnight in the presence of 5 μg/ml antibodies or control immunoglobulin G at 4°C. Immune complexes were precipitated by 50 μl of protein A or G for 2–4 h. Immunoprecipitated complexes were washed three times with Triton lysis buffer or HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol) before being boiled in 2X LDS sample buffer. 2% lysate and immnunoprecipitates were then subjected to SDS–PAGE and immunoblotting.
9
GTPase Signaling Pathway Activation
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Cells were pelleted and lysed with M-PER (Thermo Scientific Pierce) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Protein lysates were collected by centrifugation at 13,000 RPM for 10 min at 4°C and pre-cleared by incubation with Thermo Protein A/G Dynabeads (Thermo Scientific Pierce) for 3 hours at 4°C on a rotator. Pre-cleared protein lysates were quantified with the BCA Protein Assay Kit (Thermo Scientific Pierce), and 2 mg/ml lysates were prepared. We used anti-Gα12/13, anti-LARG and anti-RhoA antibodies for pull-down and immunoblotting at the recommended dilutions. Cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were scanned digitally using the ChemiDoc™ touch imaging system (Bio-Rad).
10
Comprehensive EV Protein Profiling
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Proteins were extracted from different EV populations by RIPA buffer (20 nM Tris-HCl, 150 nM NaCl, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, pH 7.8) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). The protein content was quantified by BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). Thirty micrograms of proteins were separated by electrophoresis using a 4–15% gradient sodium dodecyl sulfate–polyacrylamide gel. The proteins were transferred to a PVDF membrane by the iBlot™ Dry Blotting System (Life Technology) and then immunoblotted with the following antibodies: CD63 and CD9 (Santa Cruz Biotechnology) and hepatocyte growth factor (HGF) (Thermo Fisher Scientific). The protein bands were visualized using a ChemiDoc™ XRS+ (BioRad) with an enhanced chemiluminescence detection kit (ECL) (GE Healthcare, Amersham).
To further analyze the protein content of the different EV populations, 100 μg of EV protein was hybridized on glass slides from the Human Cytokine Array kit (Ray Biotech) according to the manufacturer's protocol. The kit allows detecting the expression of 80 different cytokines and growth factors.
To further analyze the protein content of the different EV populations, 100 μg of EV protein was hybridized on glass slides from the Human Cytokine Array kit (Ray Biotech) according to the manufacturer's protocol. The kit allows detecting the expression of 80 different cytokines and growth factors.
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