Human MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS), Penicillin-streptomycin Cocktails and Cystatin C (Catalog number: PHP0044) were obtained from Thermo Scientific (Rockford, IL). SAHA was purchased from Sigma-Aldrich (St. Louis, MO). Muse Cell Cycle kit, Muse Annexin & Dead Cell kit, and Muse Count & Viability kit were from Millipore (Darmstadt, Germany). Human MAPK Antibody Array kit was purchased from R&D Systems (Minneapolis, MN). High Pure RNA Isolation kit and Transcriptor First Strand cDNA Synthesis kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Exprofile Human autophagy Gene qPCR Array kit was obtained from Genecopoeia (Rockville, MD). Power SYBR Green PCR Master mix, RIPA Cell Lysis buffer and BCA Protein Assay kit were from Life Technologies (Austin, TX). Polyclonal anti-beclin-1 antibody, polyclonal anti-Atg3 antibody, polyclonal anti-Atg5 antibody, polyclonal anti-Atg7 antibody, polyclonal anti-Atg12 antibody, polyclonal anti-Atg16 antibody, polyclonal anti-Atg4A antibody, polyclonal anti-Atg4B antibody, polyclonal anti-Atg9B antibody, polyclonal anti-LC3II antibody were obtained from Abcam Inc (Cambridge, MA). Protease inhibitor and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Ripa cell lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
RIPA cell lysis buffer is a reagent used for the extraction and solubilization of proteins from cell and tissue samples. It is a detergent-based buffer that disrupts cell membranes and releases intracellular proteins, making them available for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Muse count viability kit, by Merck Group (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
High pure rna isolation kit, by Roche (3 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (3 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (3 mentions)
Muse cell cycle kit, by Merck Group (2 mentions)
Penicillin streptomycin cocktail, by Thermo Fisher Scientific (2 mentions)
Muse annexin dead cell kit, by Merck Group (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Human mda mb 231, by American Type Culture Collection (2 mentions)
Lamin b1, by Proteintech (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
N6 methyladenosinem6a, by Epigentek (2 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor, by Merck Group (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
48 protocols using ripa cell lysis buffer
1
Autophagy Regulation in Breast Cancer Cells
2
Chidamide Inhibits Breast Cancer Cells
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Human MCF-7 or MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS) and, Penicillin–streptomycin Cocktails were purchased from Life Technologies (Austin, TX). Chidamide was supplied by Sigma-Aldrich (St. Louis, MO). Luciferase Assay System and CellTiter 96® AQueous One Solution Cell Proliferation assay were from Promega (Madison, MI). Muse Count & Viability Kit was from Millipore (Darmstadt, Germany). High Pure RNA Isolation Kit and Transcriptor First Strand cDNA Synthesis Kit were given from Roche Diagnostics GmbH (Mannheim, Germany). 5’RACE System for Rapid Amplification of cDNA Ends, LightShift Chemiluminescent RNA EMSA Kit, Power SYBR Green PCR Master Mix, RIPA Cell Lysis Buffer and BCA Protein Assay Kit were from Life Technologies (Austin, TX). Polyclonal anti-Nestin antibody and polyclonal anti-β actin antibody were obtained from Abcam Inc. (Cambridge, MA). LncRNA ENST869 siRNA candidates were designed and synthesized by RiboBio (Guangzhou, China). Nestin siRNA(h) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other chemicals were from Sangon Biotech (Shanghai, China).
3
Protein Extraction from Cybrid Cells
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For protein extraction, normal and AMD cybrid cells were plated in 6-well plates at a density of 500,000 cells /well and were allowed to grow for 72 hr. Then, cell culture medium was removed and RIPA cell lysis buffer (Catalog no. 89900, Life Technologies) containing protease inhibitor cocktail was added to each well of the 6-well plate, and cells were scraped and transferred to microfuge tubes. Cell lysate was then centrifuged for 15 minutes at 13,000 rpm. Supernatant was transferred to a new microfuge tube and was mixed well and 10 μL of supernatant aliquoted to another tube for protein measurement. The concentration of proteins was measured using Bio-Rad Dc protein assay system (Bio-Rad Laboratories, Richmond, CA) according to the manufacturer's instructions.
4
Apoptosis Evaluation in Breast Cancer Cells
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Human MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS) and Penicillin-streptomycin Cocktails were obtained from Thermo Scientific (Rockford, IL). Suberanilohydroxamic acid (SAHA) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human TRAIL/Apo2 Ligand was from Peprotech (Rocky Hill, NJ). Muse Cell Cycle kit, Muse Annexin & Dead Cell kit, and Muse Count & Viability kit were from Millipore (Darmstadt, Germany). Human Apoptosis Antibody Array kit was purchased from R&D Systems (Minneapolis, MN). High Pure RNA Isolation kit, Annexin-V-FLUOS staining kit and Transcriptor First Strand cDNA Synthesis kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Exprofile Human Cell Cycle Tox and Cancer Related Gene qPCR Array kit and Exprofile Human EGF/PDGF Signaling Related Gene qPCR Array kit were obtained from Genecopoeia (Rockville, MD). Power SYBR Green PCR Master mix, Calcein-AM dye, RIPA Cell Lysis buffer and BCA Protein Assay kit were from Life Technologies (Austin, TX). CellTiter 96AQueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI). Protease inhibitor and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
5
Western Blot Analysis of N6-methyladenosine and FTO
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Protein lysates were prepared using RIPA Cell Lysis Buffer (cat# J63324, Alfa Aesar) supplemented with phosphatase inhibitor (cat# P5726, Millipore Sigma) and Halt™ protease inhibitor (cat# 87786, ThermoFisher Scientific) cocktails according to the manufacturers’ instructions. Equal amounts of proteins were separated on 4-12% Mini-PROTEAN TGX stain-free protein gels (cat# 4568093, Bio-Rad Laboratories) and transferred to polyvinylidene difluoride (PVDF) membranes using Trans-Blot Turbo transfer system (Bio-Rad Laboratories). The blots were incubated overnight at 4°C with primary antibodies against FTO (cat# 27226-1-AP, Proteintech), N6-methyladenosinem6A (cat# A-1801-100, EpiGenTek), Lamin B1 (cat# 12987-1-AP, Proteintech), GAPDH (cat# 60004-1-Ig, Proteintech), and β-Tubulin (cat# 10094-1-AP, Proteintech). The blots were washed and then incubated with HRP-conjugated secondary antibodies against mouse (cat# SA00001-1, Proteintech) and rabbit (cat# SA00001-2, Proteintech) at room temperature for 1 hour according to the manufacturer’s instructions. The blots were developed by an enhanced chemiluminescence (Pierce) detection system, and images were acquired using a ChemiDoc™ Touch Imaging System (Bio-Rad, USA). Densitometric analyses were performed using ImageJ (NIH) software.
6
Western Blot Analysis of N6-methyladenosine and FTO
7
Biodistribution of Fluorescent Probe
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Biodistribution was performed as previously described (24 (link)). Either 24 or 48 hours after tail vein injection with fluorescent hRS7, mice were euthanized and organs were then resected and mechanically disrupted. A solution of 5 mg/mL collagenase IV (Worthington Biochemical) in 1X RIPA cell lysis buffer (Fisher Scientific) was then added to the organs for a 1-hour incubation. Organs were sonicated using an FB-120 Sonic Dismembrator and incubated for another hour after adding 1X RIPA buffer with 0.025% trypsin-EDTA. Cells were sonicated again to complete tissue homogenization, then serially diluted and scanned on the Odyssey CLx Scanner. A calibration curve of known concentration was used to calculate the concentration of fluorescent probe, which was then normalized to organ weight and dose to calculate the %ID/g.
8
Biodistribution of Fluorescent Probe
9
Investigating SOX11 and FAK Interaction in AT2 Cells
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The transfected AT2 cells were solubilized in RIPA cell lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail and PMSF on ice for 15 min. Lysates were centrifuged at 14,000 × g for 15 min at 4°C. To assess the interaction between SOX11 and FAK, the supernatant was incubated overnight at 4°C with SOX11 antibody (Abcam; cat. no. ab170916). Subsequently, 40 µl protein A cross-linked to agarose beads (EMD Millipore; cat. no. IP05) were added, and the mixture was incubated for 1 h at 4°C with constant rotation. The beads were washed 6 times with ice-cold lysis buffer prior to the addition of SDS-PAGE sample buffer to each sample. The bound proteins were dissociated from the beads by heating at 92°C for 3 min before they were resolved on 10% SDS-gels using SDS-PAGE as described above. Western blot analysis was used to evaluate the expression of SOX11 and FAK as described above.
10
Corilagin Modulates Proteasome Subunits in U251 Cells
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Corilagin (0, 25, 50 and 100 µg/ml) was added to U251 cells (30–40% density) at 37°C which were inoculated in 6-well plates. After 48 h, proteins were extracted. Protein concentration was determined using BCA protein assay kit. U251 cells were collected and lysed using RIPA cell lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Following two washes with PBS, cells were lysed with lysis buffer [20 mM Tris-HCl (pH, 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40] containing Complete Protease Inhibitor Cocktail (Roche Applied Science). The cell lysates (40 µg) were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated with primary antibodies at a dilution of 1:1,000 overnight at 4°C. Antibodies against the following proteins were used: β1, β2, β5, β1i, β2i, β5i and β-actin. Luminata Forte Western HRP Substrate (EMD Millipore) was used for the development of positive signals. The relative protein expression of genes was quantified by ImageJ v.1.46r software (National Institutes of Health) using β-actin as a control.
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