RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, and Dulbecco's Phosphate Buffered Saline (DPBS) were purchased from Gibco-BRL (Burlington, Ont, Canada). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33342, ribonuclease A (RNase A), and propidium iodide (PI) were obtained from Sigma (St. Louis, MO, USA). DNA ladder size markers were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against Bcl-2, Bax, caspases 3 and 9, cleaved PARP, and β-actin were purchased from Cell Signaling Technology (Bedford, Massachusetts, USA). Protein marker was purchased from Bio-Rad (Richmond, CA, USA). Caspase 3 activity assay kit was purchased from Promega (San Luis Obispo, CA, USA). All other chemicals and reagents used herein were of analytical grade.
Ribonuclease a rnase a
Manufactured by Merck Group
Sourced in United States, Germany
Ribonuclease A (RNase A) is a lab equipment product used for the digestion of single-stranded RNA. It is a small, stable, and highly active enzyme that selectively catalyzes the hydrolysis of the phosphodiester bonds in RNA, resulting in the breakdown of the RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (15 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Anti β actin antibody, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Penicillin g, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Dulbecco s modified eagle s medium, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (2 mentions)
P erk, by Santa Cruz Biotechnology (2 mentions)
Sulforhodamine b, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Trichloroacetic acid, by Merck Group (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
31 protocols using ribonuclease a rnase a
1
Evaluation of Apoptosis Induction in Cell Lines
2
Immunoblotting of Histone Modifiers
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The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology. RNA Polymerase II H5 and H14 antibodies which recognize phosphoserine 2 and phosphoserine 5 versions of Pol II were from Covance. HEXIM1 sheep (ab28016) and rabbit (ab25388) polyclonal antibodies were purchased from Abcam. Anti-HDAC1 (SA-401), anti-HDAC2 (SA-402) and anti-HDAC3 (SA-403) rabbit polyclonal antibodies were obtained from BIOMOL (currently Enzo Life Sciences). Anti-SMRT (PA1–842) and anti-N-CoR (PA1–844A) antibodies were purchased from Affinity BioReagents. Anti-goat Alexa Fluor 488 (A21467) and Alexa Fluor 568 (A11057), anti-rabbit Alexa Fluor 546 (A11010) and anti-mouse Alexa Fluor 633 (A21052) IgGs, as well as TO-PRO-3 and SYTO 16 fluorescent nucleic acid stains, were obtained from Molecular Probes. Anti-acetyl-Lysine clone 4G12 mouse monoclonal IgG was from Upstate. Trichostatin A, hexamethylene bis(acetamide) (HMBA) and Ribonuclease A (RNase A) were purchased from Sigma-Aldrich.
3
Cellular Signaling Pathway Analysis
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DMEM and F12 medium, fetal bovine serum (FBS), trypan blue, penicillin G, and streptomycin were obtained from Invitrogen (Gaithersburg, MD, USA). Dimethyl sulphoxide (DMSO), ribonuclease A (RNase A), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against JNK, p38 (sc-7149), p-p38 (Tyr182, sc-7973), ERK, p-ERK (Tyr204, sc-7976), COX-2, and β-actin (sc-7963) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against SP-1 (5407-S) was purchased from Epitomics. Antibody against p-JNK (Thr183/Tyr185, #07-175) was purchased from Millipore. Anti-rabbit, anti-goat and anti-mouse IgG peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL, USA). Annexin V-FITC staining kit was purchased from Strong Biotech Co. Ltd. (Taipei, Taiwan).
4
Cell Culture and Protein Expression Analysis
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
phosphate buffer saline (PBS), penicillin and streptomycin were
obtained from Hyclone (Logan, UT, USA).
Lipofectamine®RNAiMAX and TRIzol® Reagent was purchased
from Invitrogen (Carlsbad, CA, USA). Ribonuclease A (RNase A) and
DNAse were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
antibodies used were the following: rabbit anti-Inhibin beta B
polyclonal antibody (17577-1-AP, 1:200) purchased from Proteintech
Group (Chicago, IL, USA); anti-bax (BS-2538, 1:800) purchased from
Bioworld Technology (St. Louis Park, MN, USA); anti-cyclin D1 (sc-753,
1:200), anti-cyclin E (sc-481, 1:200), anti-Bcl-2 (sc-783, 1:200 and
anti-GAPDH (sc-59540, 1:3,000) ) purchased from the Santa Cruz
Biotechnology (Dallas, TX, USA).
phosphate buffer saline (PBS), penicillin and streptomycin were
obtained from Hyclone (Logan, UT, USA).
Lipofectamine®RNAiMAX and TRIzol® Reagent was purchased
from Invitrogen (Carlsbad, CA, USA). Ribonuclease A (RNase A) and
DNAse were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
antibodies used were the following: rabbit anti-Inhibin beta B
polyclonal antibody (17577-1-AP, 1:200) purchased from Proteintech
Group (Chicago, IL, USA); anti-bax (BS-2538, 1:800) purchased from
Bioworld Technology (St. Louis Park, MN, USA); anti-cyclin D1 (sc-753,
1:200), anti-cyclin E (sc-481, 1:200), anti-Bcl-2 (sc-783, 1:200 and
anti-GAPDH (sc-59540, 1:3,000) ) purchased from the Santa Cruz
Biotechnology (Dallas, TX, USA).
5
Apoptosis Assay in Cell Lines
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Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), trypsin, monodansylcadaverine (MDC) stain, propidium iodide (PI), ribonuclease A (RNase A) and all other additional chemicals not specifically mentioned were of analytical grade and were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Penicillin, streptomycin and fungizone were bought from Thermo Fisher Scientific (Waltham, MA, USA), and fetal calf serum (FCS) was purchased from Biochrom GmbH (Berlin, Germany). Crystal violet, 1% glutaraldehyde and Triton X-100 were purchased from EMD Millipore (Billerica, MA, USA). Ethics approval was obtained from The Research Ethics Committee, Faculty of Health Sciences, University of Pretoria, South Africa (reference number: 258/2015).
6
Cell Cycle Analysis by Flow Cytometry
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WERI-Rb1 cells or Y79 cells were harvested, fixed with 75% ice-cold ethanol in PBS and kept at 4°C. Prior to analysis, cells were washed twice with PBS and then incubated for 30 min in a propidium iodide staining solution containing 0.05 mg/ml propidium iodide, 1 mM ethylene-diaminetetraacetic acid (EDTA), 0.1% Triton X-100™ and 1 mg/ml ribonuclease A (RNase A) (all from Sigma-Aldrich, St. Louis, Missouri). The staining fluorescence intensity was measured using a cytomics FC500 MCL flow cytometer (Beckman Coulter Inc, USA) and used to determine the G1/M ratio.
7
DNA Damage Response Inhibitor Assay
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Calf intestine alkaline phosphatase, micrococcal nuclease, calf spleen phosphodiesterase and ribonuclease A (RNase A) were purchased from Sigma (Steinheim, Germany). Proteinase K, HPLC-grade methanol, formic acid and acetic acid were from Carl Roth GmbH (Karlsruhe, Germany). The synthesis of the isotope-labeled reference standard [15N5,13C10]C8-PhIP-dG was previously described (9 (link)). The CHK1 inhibitor UCN-01 was obtained from Sigma. The ATR inhibitor VE821, the ATM inhibitor KU-55933 and the DNA-PKcs inhibitor NU7026 were from Selleck Chemicals (USA).
8
Antiparasitic Drug Screening Assay
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Fetal bovine serum (FBS) and RPMI 1640 media, sodium pyruvate, penicillin-streptomycin and L-glutamine, were purchased from Invitrogen Gibco, Incl. LIT, RPMI 1640-modified medium and HMI-9 media were prepared at Tissue culture facility in Instituto de Parasitología y Biomedicina “López-Neyra”. Surfact-Amps NP40 was supplied by Thermo Scientific and Tween 20 by Merck. Sodium dodecyl sulfate (SDS) was purchased from Affymetrix. Chlorophenol red-β-D-galactopyranoside (CPRG), resazurin sodium salt, amphotericin B, p-formaldehyde, pentamidine isethionate salt, benznidazole, nifurtimox, posaconazole, ATP disodium salt, propidium iodide, podophyllotoxin, ribonuclease A (RNase A) from bovine pancreas, phorbol 12-myristate 13-acetate (PMA),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich. CellTiter-Glo Luminescent Cell Viability Assay was purchased from Promega Corporation and 4′, 6-diamidino-2-phenylindole (DAPI) Prolong from Molecular Probes.
9
Evodiamine Cytotoxicity Assay Protocol
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Trichloroacetic acid (TCA), sulforhodamine B (SRB), bovine serum albumin (BSA), propidium iodide (PI), ribonuclease A (RNase A), and anti-β-actin antibody were purchased from Sigma (St.Louis, MO, USA). RPMI 1640, fetal bovine serum (FBS), trypsin-EDTA and antibiotic-antimycotic solution were purchased from GIBCO-BRL (Grand Island, NY, USA). Antibodies against Cdk4, Cdk2, Cyclin A, Cyclin B1, Bcl-2, Bax, p-ERK, ERK and CDK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-cdc2 (Tyr15), p-Chk1 (Ser345), p-Chk2 (Thr68), Chk1, Chk2, p-H2A.X (Ser139), procaspase-3, caspase-8, LC3B and Beclin-1 were obtained from Cell Signaling (Danvers, MA, USA). The test compounds including evodiamine (Fig. 1 ) were isolated by Dr. Jung Joon Lee (Korea Research Institute of Bioscience and Biotechnology, Korea) from Evodia rutaecarpa Bentham (Rutaceae) and provided the compounds through the Research Center for Standardization of Herbal Medicines in Korea.
10
Chemotherapeutic Drug Solubilization Protocol
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Sorafenib tosylate (Bay 54-9085) (hereafter referred to as sorafenib) was purchased from Bayer HealthCare (Bayer Schering Pharma, Berlin, Germany) and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 1 mM and stored at − 80°C. U0126 (Mek1/2 inhibitor) (New England Biolabs, Frankfurt, Germany) was dissolved in DMSO as 10 mM stock concentration and stored at − 20°C. Propidium iodide (PI) (Sigma-Aldrich, Taufkirchen, Germany) was prepared as 0.5 mg/mL stock concentration in phosphate-buffered saline (PBS) and stored at 4°C. Ribonuclease A (RNase A; Sigma-Aldrich; stock solution 1 mg/mL) and recombinant murine interleukin-3 (rm IL-3) (Immuno Tools, Friesoythe, Germany; stock concentration 50 μg/mL) were prepared in water and stored at − 20°C. Cytarabine (CellPharm, Hannover, Germany) was stored as 412 mM stock solution in 0.9% sodium chloride.
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