Treated cells were lysed by ice-cold cell lysis buffer (Intron Biotechnology, Seongnam, Korea), and protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. Equal amounts of protein (20–30 μg/lane) was separated on a 12% acrylamide gel by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blocked with 5% non-fat milk. Each membrane was incubated with anti-Chk1, anti–phospho Chk1 (Ser345), anti–phospho Chk1 (Ser296), anti-PARP, anti–cyclin B1 (Cell Signaling Technology, Danvers, MA); anti-Rad51, anti–γ-H2AX (Ser139), anti–phospho histone H3 (Ser10), anti–cleaved caspase-3, anti–cleaved PARP (Abcam, Cambridge, UK); and alpha-tubulin (Sigma-Aldrich) antibodies. Each membrane was then incubated with horse radish peroxidase (HRP)–conjugated secondary anti-mouse or rabbit IgG antibody, and protein bands visualized using Immobilin Forte Western HRP Substrate (Millipore, Burlington, MA).
Cell lysis buffer
Manufactured by iNtRON Biotechnology
Sourced in United States
Cell lysis buffer is a reagent used to disrupt the cell membrane and release the cellular contents, including proteins, nucleic acids, and other biomolecules. It is a crucial component in various molecular biology and biochemistry techniques, such as protein extraction, DNA/RNA isolation, and cell fractionation.
Automatically generated - may contain errors
Lab products found in correlation
Alpha tubulin, by Merck Group (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Merck Group (2 mentions)
Anti cyclin b1, by Cell Signaling Technology (1 mentions)
Anti rad51, by Abcam (1 mentions)
Anti phospho chk1, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Anti phospho histone h3, by Abcam (1 mentions)
Anti cleaved parp, by Abcam (1 mentions)
Anti γh2ax, by Abcam (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Secondary antibodies linked to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Fusion capt advance software, by Vilber (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
9 protocols using cell lysis buffer
1
Protein Expression Analysis Protocol
2
C2C12 Cell Western Blot for Akt and Foxo Signaling
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C2C12 cells were prepared for western blotting to detect phospho-Akt, total Akt, and β-actin expression. The cells were washed with cold PBS twice before harvesting. Cells were harvested in 150 μl of cell lysis buffer purchased from iNTRON Biotechnology Inc. (Daejeon, Korea). After collecting the cells, the samples were boiled for 10 min in 95 °C. After boiling, samples were centrifuged at 13,000 rpm for 10 min at 4 °C. The concentration of collected supernatants was determined by a BCA assay kit (Thermo Scientific).
The protein samples (30 μg) were loaded on the 8–10% polyacrylamide gel and blotted on a PVDF membrane in transfer buffer at 200 mA, 2 h after electrophoresis. The blots were blocked with 10% milk in PBST (0.1% Tween-20 in PBS) for 1 h, at room temperature. The blots were incubated with antibodies specific to phospho-Akt (1:1000), Akt(1:1000), phospho-Foxo(1:1000), Foxo(1:1000) and β-actin (1:2000), overnight at 4 °C. The blots were washed three times using PBST, and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10000) for 1 h, at room temperature. After incubation, the blots were washed again with PBST. After washing, the blots were detected with SuperSignal West Pico or West Femto Chemiluminescent Substrate, using X-ray film.
The protein samples (30 μg) were loaded on the 8–10% polyacrylamide gel and blotted on a PVDF membrane in transfer buffer at 200 mA, 2 h after electrophoresis. The blots were blocked with 10% milk in PBST (0.1% Tween-20 in PBS) for 1 h, at room temperature. The blots were incubated with antibodies specific to phospho-Akt (1:1000), Akt(1:1000), phospho-Foxo(1:1000), Foxo(1:1000) and β-actin (1:2000), overnight at 4 °C. The blots were washed three times using PBST, and then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10000) for 1 h, at room temperature. After incubation, the blots were washed again with PBST. After washing, the blots were detected with SuperSignal West Pico or West Femto Chemiluminescent Substrate, using X-ray film.
3
Western Blot Analysis of Proteins
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Cultured cells were extracted and lysed for 60 min at 4 °C in cell lysis buffer (Intron Biotechnology, Seoul, Republic of Korea). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on equal amounts of proteins (30–40 μg) using 10% gel. The extracted proteins were separated and put to a nitrocellulose membrane (Amersham Bioscience, Westborough, MA, USA). The transferred membranes were probed with the corresponding primary antibodies, followed by binding with secondary antibodies linked to horseradish peroxidase (Santa Cruz Biotechnology). An enhanced chemiluminescence kit was used to detect immunoreactive signals (Amersham Bioscience).
4
Western Blot Analysis of Protein Expression
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The cells were collected, washed twice with ice-cold phosphate-buffered saline (PBS), and then lysed with ice-cold cell lysis buffer (iNtRON Biotechnology, Gyeonggi-do, South Korea) containing freshly added protease and a phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA, USA) on ice for 10 min. Samples were centrifuged at 13,000× g for 10 min at 4 °C. A sodium dodecyl sulfate (SDS) loading buffer was added to the supernatants, with subsequent incubation for 10 min at 100 °C. Aliquots of 20 μg of proteins were separated by SDS–polyacrylamide gel electrophoresis and electroblotted to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk in PBS containing 0.05% Tween 20 (PBS-T) and then incubated overnight at 4 °C with indicated primary antibodies. After washed thrice with PBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, followed by PBS-T wash three times and the bands were visualized using an ECL system (Merck, Rahway, NJ, USA). The intensities of the protein bands were quantified by ImageJ software (version 1.47). The protein expression of GAPDH was used as an internal control for normalization.
5
Western Blot Analysis of Protein Expression
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The cells were collected, washed twice with ice-cold phosphate-buffered saline (PBS), and then lysed with ice-cold cell lysis buffer (iNtRON Biotechnology, Gyeonggi-do, South Korea) containing freshly added protease and a phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA, USA) on ice for 10 min. Samples were centrifuged at 13,000× g for 10 min at 4 °C. A sodium dodecyl sulfate (SDS) loading buffer was added to the supernatants, with subsequent incubation for 10 min at 100 °C. Aliquots of 20 μg of proteins were separated by SDS–polyacrylamide gel electrophoresis and electroblotted to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk in PBS containing 0.05% Tween 20 (PBS-T) and then incubated overnight at 4 °C with indicated primary antibodies. After washed thrice with PBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, followed by PBS-T wash three times and the bands were visualized using an ECL system (Merck, Rahway, NJ, USA). The intensities of the protein bands were quantified by ImageJ software (version 1.47). The protein expression of GAPDH was used as an internal control for normalization.
6
Tranylcypromine Modulates LPS-induced ERK Signaling
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The effects of tranylcypromine on LPS-induced ERK signaling were assessed in BV2 microglial cells treated successively with LPS (1 μg/mL) or PBS for 45 min and tranylcypromine (5 μM) or vehicle (1% DMSO) for 45 min. The cells were then prepared for western blotting by lysis in Cell Lysis Buffer (ProPrep, iNtRON Biotechnology, Inc., Seongnam, Korea). After centrifuging the lysate at 12,000 rpm for 15 min, the supernatant was collected. The protein samples were separated by SDS gel electrophoresis and then, electrotransferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked with 5% skim milk or 5% BSA and incubated with anti-ERK (1:1000, Santa Cruz Biotechnology) or anti-p-ERK (1:1000, Cell Signaling) antibodies overnight. After incubating the membranes with horseradish peroxidase-conjugated secondary antibody for 1 h, detection was achieved with ECL Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). Fusion Capt Advance software (Vilber Lourmat) was used to acquire and analyze images.
7
Anti-inflammatory Effects of Aster incisus
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Human kidney cells (HEK293 cells), human keratinocytes (HaCaT cells), and murine macrophages (RAW 264.7 cells) were from the ATCC (Manassas, VA, USA). DMEM media were obtained from Hyclone Laboratories (USA), and fetal bovine serum (FBS) and penicillin-streptomycin were from Cellgro (Manassas, VA, USA). The Aster incisus methanol extract (voucher no. 016-001) was purchased from the Korean Plant Extract Bank (KPEB, Cheongju, Korea). EZ-Cytox (WST-1; Daeil Lab Service, Seoul, Korea), dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA), LPS (Sigma, MO), Griess reagent (Sigma, USA), and cell lysis buffer were purchased from Intron Biotechnology Inc., Gyeonggi, Korea. The first antibodies (iNOS, Cox-2, TNFα, pIκBα, IκBα, p-p65 NFκB, NFκB, p-Akt, p-PI3K, p-mTOR, p-SAPK/JNK, p-ERK1/2, and p-p38) and the second antibody linked to a peroxidase were purchased from Cell Signaling Technology (CST; Danvers, MA, USA). ECL detection solution was obtained from Pierce (Rockford, IL, USA) and 4% formaldehyde from Sigma (Sigma-Aldrich, St. Louis, MO, USA) while rabbit normal serum and anti-rabbit IgG were purchased from Cell Signaling Technology (CST; Danvers, MA, USA).
8
Nobiletin Cytotoxicity in U2OS and HOS Cells
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We plated 6×105 U2OS and HOS cells in 6 cm plates for 16 h, followed by the treatment with different concentrations (0, 25, 50, 75, 100 μM) of nobiletin for 24 h. Nuclear extracts were obtained as Sigma-Aldrich procedure described. The cells were lysed with ice-cold buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCL, 1 mM DTT, 0.5 mM PMSF, plus protease and phosphatase inhibitors), followed by dounce homogeniser and microcentrifuge to shear the cytoplasmic membranes. Cytoplasmin extracts were on the supernatant, resuspended pellet in cell lysis buffer (iNtRON Biotechnology, INC), and pipetted up and down to achieve homogenous mix. Followed by centrifugation at 13000 rpm for 30 min at 4°C in a microcentrifuge, we harvested the nuclear extract from the supernatant and stored at −20°C. The total and extracts protein were determined by BCA assay.
9
Protein Expression Analysis in Cell Lysates
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Cells were lysed by ice-cold cell lysis buffer (Cat# 17081, Intron Biotechnology, Seongnam, Korea) with the protease inhibitor cocktail. Protein concentrations were determined with a BCA assay kit (Cat# 23117, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Protein was separated via 10% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% non-fat milk. Membranes were incubated with anti-HOXB9 (Cat# PA5-101087, Thermo Fisher Scientific, Waltham, MA, USA), anti-ERCC-1 (Cat# ab129267), anti-MRP-2 (Cat# ab172630), anti-Bcl-2 (Cat# ab182858, abCAM, Cambridge, UK), anti-XIAP (Cat# 14334), anti-MMP-2 (Cat# 40994), anti-Bax (Cat# 5023), anti-cleaved caspase-3 (Cat# 9661), anti-cleaved PARP (Cat# 5625, Cell Signaling Technology, Inc., Danvers, MA, USA), and alpha-tubulin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Next, the membranes were incubated with HRP-conjugated anti-secondary Mouse IgG antibody (Cat# 7076) or HRP-conjugated anti-secondary Rabbit IgG antibody (Cat# 7074, Cell Signaling Technology, Inc., Danvers, MA, USA), and the visualized using Immobilin Forte Western HRP Substrate (Cat# WBLUF0100, Merk Millipore, Burlington, MA, USA).
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