Proteins from cardiac samples and H9c2 cells were extracted using a ProteoJET Mammalian Cell Lysis Reagent (Fermentas, MD, U.S.) containing protease and phosphatase inhibitors. The lysate solution was centrifuged for 15 min at 4,000 x g at 4°C. The supernatant was transferred into new tubes. After quantification of protein was performed using BCA Protein Assay, 40 μg of total protein was electrophoresed using 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, MA, U.S.). Membranes were blocked for 1 h at 4°C with 5% non-fat milk. Then they were incubated overnight at 4°C with the primary antibody (rabbit monoclonal anti-phospho-specific p38, anti-phospho-specific JNK, anti-phospho-specific ERK, anti-p38, anti-JNK, anti-ERK, anti-bcl-2, anti-cleaved caspase 3, anti-GAPDH, Cell Signaling, CA, U.S.; rabbit monoclonal anti-RAGE, anti-ADAM10, Abcam, MA, U.S.) diluted 1:1000 in TBST with 5% BSA. Membranes were washed with TBST, followed by an incubation of 1 h at room temperature with either horseradish-peroxidase-conjugated goat anti-rabbit secondary antibodies or goat anti-mouse (1:1000). The positive protein bands were developed using a chemiluminescent system and semi-quantified using Quantity One 1-D Analysis Software (Bio-Rad, CA, U.S.). GAPDH served as an internal control for normalization of protein quantity.
Proteojet mammalian cell lysis reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The ProteoJET Mammalian Cell Lysis Reagent is a solution designed for the efficient lysis of mammalian cells. It is used to extract cellular proteins for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ecl detection system, by Merck Group (5 mentions)
Gapdh, by Abcam (4 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Alpha imager, by Bio-Techne (3 mentions)
Goat anti rabbit or anti mouse igg, by Jackson ImmunoResearch (3 mentions)
1 step nbt bcip reagent, by Thermo Fisher Scientific (3 mentions)
Ab8226, by Abcam (3 mentions)
Startingblock blocking buffer, by Thermo Fisher Scientific (2 mentions)
Quantity one, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
C myc, by Abcam (2 mentions)
Phosstop, by Roche (2 mentions)
Dual color protein loading buffer, by Thermo Fisher Scientific (2 mentions)
46 protocols using proteojet mammalian cell lysis reagent
1
Proteomic Analysis of Cardiac Samples
2
Western Blot Analysis of Osteogenic Signaling
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AVICs (5×106 cells) were lysed with the ProteoJET Mammalian Cell Lysis reagent (Fermentas; Thermo Fisher Scientific, Inc.) to extract cytoplasmic proteins. Equal quantities of protein (50 µg) were subjected to 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membrane was blocked with 5% bovine serum albumin (catalog no. V900933; Sigma-Aldrich; Merck Millipore) and probed with antibodies against TLR4 (dilution, 1:500), Runx2 (dilution, 1:1000), Msx2 (dilution, 1:1,000), BMP2 (dilution, 1:1,000), OPN (dilution, 1:1,000), β-actin (dilution, 1:2,000), p38-MAPK (dilution, 1:1,000), p-p38-MAPK (dilution, 1:1,000), NF-κB (dilution, 1:1,000) and p-NF-κB (dilution, 1:1,000) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies (dilution, 1:5,000) for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection system (Merck Millipore). Images were captured, and the intensity of each band was analyzed with Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Cell Lysis and Immunoblotting Protocol
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Cell lysates were prepared using the ProteoJET Mammalian Cell lysis reagent (Fermentas) and the 1× protease inhibitor mixture (Roche Molecular Biochemicals, Pleasanton, CA). Immunoblotting was performed as described earlier [30] (link). Monoclonal antibodies against Bcl-2, GAPDH and cleaved caspase-9 (Cell Signalling, Boston, MA, USA) were used.
4
Western Blot Analysis of Protein Extracts
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Treated cells were washed in ice-cold PBS and extracted with ProteoJET™ Mammalian Cell Lysis Reagent (Fermentas, Burlington, ON, Canada) supplemented with protease and phosphatase inhibitors (Fermentas) according to the manufacturer’s instructions. Protein (20–40 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 8%–10% gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Blots were blocked for 1 hour at room temperature with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline/0.1% Tween-20. The blots were then incubated with specific primary antibodies at 4°C overnight. The blots were then incubated with horseradish peroxidase-linked secondary antibodies for 1 hour at room temperature. After additional washes, signals were detected using SuperSignal ECL (Pierce, Rockford, IL, USA).
5
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Culture cells were washed with PBS and the total protein was extracted using ProteoJET Mammalian Cell Lysis Reagent (Fermentas) according to the manufacturer’s instructions. Nuclear protein fraction was prepared using Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. In general, 20 μg total proteins were loaded onto 10% SDS-polyacrylamide gels and then transferred onto PVDF membranes after electrophoresis. The transferred membranes were blocked with StartingBlock Blocking Buffers (Thermo Scientific) for 10 min and incubated with specific primary antibody against E-cadherin (BD), Vimentin (BD), IGFBP-7, phospho T308 AKT1, AKT1, phosphor S9 GSK3β, GSK3β (Abcam), β-catenin (GeneTax), or β-Actin (Cell signaling) at 1:1000 dilutions at 4°C overnight. The membranes then were washed three times in Tris Buffered Saline with 0.1% Tween-20 (TBST). Bands were detected using a horseradish peroxidase-linked second antibody and enhanced chemiluminescence reagents, according to the manufacturer's protocol. Bands were record by film and the intensity of the band was quantified with a GS-800 calibrated densitometer (Bio-Rad), and calculated as the optical density area of bands. The data were conducted independently in triplicate.
6
Western Blot Analysis of Protein Expression
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Cell lysates were prepared with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada). Twenty µg of the cell lysate isolated from Rin-5F or islet cells or 20 µg proteins precipitated from the cell culture medium after cell treatment were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblot analysis was performed using anti-PARP-1 (H-250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (H10; Alexis Biochemicals, San Diego, CA, USA), anti-Akt 1/2/3 (H-136; Santa Cruz Biotechnology), anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology), anti-ERK 1/2 (K-23; Santa Cruz Biotechnology), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies. Blots were probed with horseradish peroxidase-conjugated secondary antibodies. Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech, Amersham, UK). If different set of samples were run on different gels, immunoblot analysis was performed in the same time, under the same conditions. All membranes were exposed under the same film and thus exposure and development did not influence the obtained results.
7
Native Protein Detection and Quantification
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Cells were lysed with ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Hanover, MD, USA), following the manufacturer’s protocol, to obtain total native conformation proteins. Protein concentrations were determined using a Bio-Rad Protein Assay Kit. A molecular weight (kDa) standard (NativeMark - Novex, Life Technologies, Grand Island, NY, USA) and equal amounts of protein extracts (50 μg) were subjected to electrophoresis on a 8 % SDS-free polyacrylamide gel (PAGE) and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane following the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). After blocking non-specific binding sites with 5 % skimmed milk, blots were incubated with primary rabbit polyclonal antibody specific to eIF3f (Biolegend, San Diego, CA, USA) or primary goat antibody specific to alpha 1B-ADR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish peroxidase-conjugated goat anti-rabbit (Biolegend) or rabbit anti-goat secondary antibody (Santa Cruz Biotechnology). The bound antibody was detected by enhanced chemiluminescence (ECL) on an X-ray film (GE Healthcare Life Sciences, Piscataway, NJ, USA).
8
Protein Expression Analysis Workflow
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Cells were lysed with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, MD, USA) to extract cytoplamic proteins. Equal amounts of protein extracts were subjected to 12% SDS/PAGE and blotted onto a poly (vinylidenedifluoride) membrane. The membrane was blocked and probed overnight at 4°C with antibodies against TLR4 (1∶500), β-actin (1∶2000), total p38 (1∶1000), phosphorylated p38 (1∶1000), total NF-κB (1∶1000), phosphorylated NF-κB (1∶1000), total ERK1/2 (1∶1000), phosphorylated ERK1/2 (1∶1000), total JNK (1∶1000),phosphorylated JNK (1∶1000), IL-1β (1∶1000), TNFα (1∶1000), MCP-1 (1∶1000), and MMP2 (1∶1000) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1∶5000) for 1 h at room temperature. Blots were developed using an ECL detection system (Millipore, MA, USA). Each image was captured and the intensity of each band was analyzed with Quantity One (Bio-Rad).
9
Protein Extraction and Western Blot Analysis
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Proteins from normal and ADPKD tissues were obtained using ProteoJET™ Mammalian Cell Lysis Reagent (Fermentas) according to manufacturer’s protocol, and then analyzed by Western blotting to detect CDKN1C (Cell Signaling Technology, 1:1000) and β-actin (Cell Signaling Technology, 1:1000). The secondary antibody was purchased from Sigma.
10
Western Blot Analysis of Pak4 and AKT
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Cells were lysed using ProteoJET Mammalian Cell Lysis Reagent (MBI Fermentas, ON, Canada) with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Total protein concentration was estimated using the BCA method (Pierce, Rockford, IL, USA). A total of 60 μg of protein was separated on an 8% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated with primary antibodies. Signal was detected using the BeyoECL Plus (Beyotime, Shanghai, PR China).
Primary antibodies were: rabbit anti-Pak4 (1:1000, Abcam, Cambridge, UK), rabbit anti-p-Pak4 Ser474 (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:2000, ProteinTech Group, Chicago, IL), rabbit anti-AKT1 (1:1000, Epitomics, Burlingame, CA, USA), and rabbit anti-p-AKT Ser473 (1:1000, Epitomics, Burlingame, CA, USA).
Primary antibodies were: rabbit anti-Pak4 (1:1000, Abcam, Cambridge, UK), rabbit anti-p-Pak4 Ser474 (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:2000, ProteinTech Group, Chicago, IL), rabbit anti-AKT1 (1:1000, Epitomics, Burlingame, CA, USA), and rabbit anti-p-AKT Ser473 (1:1000, Epitomics, Burlingame, CA, USA).
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