Cell lysates were prepared in NETN lysis buffer [0.5% NP-40 (Abcam, Cambridge, MA, USA), 1 mM EDTA (Bioneer, Daejeon, South Korea), 120 mM NaCl, 1 mM DTT (Sigma-Aldrich), 10 mM NaF (Sigma-Aldrich), 2 mM Na3VO4 (Sigma-Aldrich), 50 mM Tris-Cl, pH 8.0) with a protease inhibitor cocktail (Sigma-Aldrich). Lysates were incubated with the indicated antibodies overnight at 4°C and Protein G Sepharose beads (GE Healthcare Life Sciences) were added. Following incubation for 2 h, the immune complexes were washed and released from the beads by boiling and then analyzed by western blotting using the indicated antibodies.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Bioneer
Sourced in United States
EDTA is a laboratory chemical used as a chelating agent. It binds to metal ions, including calcium, magnesium, and heavy metals, forming stable complexes. EDTA is commonly used in analytical procedures, such as titrations and separations, to control the presence of specific ions.
Automatically generated - may contain errors
Lab products found in correlation
Np 40, by Abcam (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Na3vo4, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hiseq 2500 instrument, by Illumina (1 mentions)
Truseq mrna sample prep kit, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Isoamyl alcohol, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Bioneer (1 mentions)
Phenol chloroform, by Merck Group (1 mentions)
Sodiumacetate, by Bioneer (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Nanodrop one uv spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Human complement serum, by Innovative Research (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
5 protocols using ethylene diamine tetraacetic acid (edta)
1
Immunoprecipitation and Western Blotting
2
Total RNA Extraction and RNA-seq Library Preparation
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Total RNA was extracted with some modification from a previous method17 (link). Briefly, the cells were washed with the extraction buffer of 200 mM Tris-HCl pH 7.5 (Bioneer, Daejeon, Korea republic of), 25 mM EDTA (Bioneer), 250 mM NaCl (Bioneer), and 0.5% SDS (Sigma-Aldrich, St. Louis, MO, USA), which was instantly frozen using liquid nitrogen and ground into a find powder by using a pestle and mortar. The pulverized cells were immediately transferred to TRIzol reagent (Invitrogen, CA, USA) and the total RNA was purified using ethanol precipitation. The RNA-seq library was constructed using the TruSeq mRNA Sample Prep Kit (Illumina, CA, USA) according to the manufacturer’s instructions. Briefly, poly-A tailed mRNA was purified with provided oligo dT magnetic beads and fragmented for 2 min to obtain RNA fragments longer than 150 bp. cDNA was synthesized from the fragmented RNA and A-tailed to both ends of the cDNA. The A-tailed cDNA was ligated to adaptors and the properly ligated products were then enriched by polymerase chain reaction (PCR). The resulting libraries were sequenced by the HiSeq 2500 instrument using the 100 bp single-end recipe (Illumina).
3
Rat Ovary Genomic DNA Extraction
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Genomic DNA (gDNA) was extracted from the rat ovaries via treatment with proteinase K (QIAGEN, Valencia, CA, USA) and phenol/chloroform (Sigma-Aldrich). The ovary tissues of rats were ground in LN2, and the powdered tissue was then digested overnight at 55 °C in digestion buffer [100 mM Tris pH 8.0 (Abelbio, Jungnang, Seoul, Korea) containing 5 mM EDTA (Bioneer, Daedeok, Daejeon, Korea), 0.2% sodium dodecyl sulfate (SDS; Bioneer), 200 mM sodium chloride (Bioneer), and 0.5 mg/ml proteinase K (Dako, Cambridge, U.K.)]. The supernatants containing gDNA were extracted by using phenol/chloroform (1:1, Sigma-Aldrich) and precipitated with isoamyl alcohol (Sigma-Aldrich) and 0.3 M sodium acetate (Bioneer) at − 20 °C overnight. Next, the gDNA pellet was washed with cold 70% ethanol and eluted using a Tris-EDTA buffer. The gDNA for each sample was analyzed using 1% agarose gel electrophoresis.
4
Self-Assembling DNA Nanostructures with caDNAno
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Using an open source program, caDNAno53 (link), all structures used here were designed on the honeycomb lattice with a M13mp18 scaffold strand (7249-nt-long, GUILD, www.guildbioscience.com ). Sequences of staple strands for the structures were exported from the caDNAno (Supplementary Tables 1 –5 ) and they were synthesized from Bioneer (www.bioneer.co.kr ). A folding mixture consists of 20 nM concentration of scaffold DNA, 100 nM concentration of each staple strand, 1 × TAE buffer (40 mM Tris-acetate and 1 mM EDTA, Bioneer) and 20 mM of MgCl2 (Sigma-Aldrich, www.sigmaaldrich.com ). The annealing process for self-assembly of DNA strands was performed by establishing temperature gradients from 80 to 60 °C with a rate of −0.25 °C/min and from 60 to 45 °C at a rate of −1 °C/hr in a thermocycler (T100, Bio-Rad, www.bio-rad.com ). Excessive staple strands were removed through five buffer exchange procedures35 (link) at 5 krcf during 8 min and concentration of structures was adjusted using the same buffer used in folding (1 × TAE and 20 mM of MgCl2). Concentrations of folded structures were measured using a Nanodrop One UV spectrophotometer (Thermo Fisher Scientific, www.thermofisher.com ). Purified structures were stored at −4 °C in a refrigerator.
5
Pig Blood Cells Viability Assay
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2 × 105 of PBMCs and RBCs isolated from pigs were incubated with 0%, 3.125%, 6.25%, 12.5%, and 25% of pooled human complement serum (Innovative research, USA) for 2 h (PBMCs) or overnight (RBCs) on v-bottom 96-well plates (Corning, USA). After incubation, human complement serum was removed following centrifugation. PBMCs were then re-suspended with 100 µL of RPMI 1640 media (Gibco) containing 5% fetal bovine serum (Gibco), 15 mM HEPES (Sigma), and 0.2 M EDTA (Bioneer, Korea). To estimate viability against human complement serum, 10 µL of CCK-8 solution was added to suspended PBMCs, and the absorbance of each well at 450 nm was measured. After incubation of RBCs with human complement serum, viability was estimated by measurement of the absorbance (405 nm) of the remaining viable RBCs.
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