The isolation of total protein from tissues and cells was performed using RIPA lysis buffer (Beyotime, Shanghai, China), followed by quantification analysis using the BCA kit (Beyotime). Subsequently, protein samples (30 μg/lane) were separated through sodium dodecyl sulphate–polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane (Millipore). Then, the membrane was sealed with 5% skim milk for 1 h, subjected to incubation with HSF1 (4356, CST), Vimentin (ab92547, Abcam), E-cadherin (14472, CST), N-Cadherin (13116, CST), and GAPDH (5174, CST) overnight and horseradish peroxidase-labeled IgG (ab124055, Abcam) for 1 h, and developed with ECL kits (Thermo Fisher Science) in a chemiluminescent imager (Image Quant LAS4000 mini, GE Healthcare, UK). The Image J software analyzed protein bands.
Ab124055
Manufactured by Abcam
Sourced in United Kingdom
Ab124055 is a laboratory antibody product. It is a reagent for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Water bath sonicator, by Qsonica (2 mentions)
Total h3, by Cell Signaling Technology (2 mentions)
H3k9ac, by Active Motif (2 mentions)
Magna chip g tissue kit, by Merck Group (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
H3k4me3, by Cell Signaling Technology (2 mentions)
H3k9me2, by Abcam (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Ab92547, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab62344, by Abcam (1 mentions)
Ab243142, by Abcam (1 mentions)
Ab193262, by Abcam (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Polyattract mrna isolation systems, by A&D Company (1 mentions)
5 protocols using ab124055
1
Protein Isolation and Western Blot Analysis
2
Chromatin Immunoprecipitation from Rat DRG Tissue
3
Chromatin Immunoprecipitation from Rat DRG Tissue
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We performed chromatin immunoprecipitation (ChIP) using the Magna ChIP G tissue kit (Millipore; catalog #17-20000), according to the manufacturer’s instructions. Briefly, fresh DRG tissues were rapidly removed from anesthetized rats and were stabilized for 3 min using stabilization buffer. Then, the DRGs were incubated in 2% formaldehyde for 20 min at ~26 °C. After being washed three times with PBS, the DRGs were incubated in lysis buffer for 15 min on ice. Finally, the DRG tissues were sonicated (30 s on and 30 s off, repeated 200 times) in ChIP dilution buffer using a water bath sonicator (Qsonica, Newtown, CT) at 4 °C. The sonicated DRG samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Chromatin was pulled down using the following antibodies: IgG (as a negative control; catalog #ab124055, Abcam), total H3 (catalog #2650s, Cell Signaling Technology), H3K9me2 (catalog #ab1220, Abcam), H3K9ac (catalog #39917, Active Motif), H3K27me3 (catalog #9773s, Cell Signaling Technology), and H3K4me3 (catalog #9751s, Cell Signaling Technology). After chromatin precipitation, we performed quantitative PCR using the primers described in Supplementary Table 4 . Data were analyzed and corrected by input and total H3 conditions 60 (link).
4
RIPK3-Mediated Necroptosis Signaling Pathway
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INS-1 cells overexpressing RIPK3 were grown in T75 flasks, treated with TNFα+zVAD for 24 h, and lysed in buffer containing 20 mM Tris HCl, 137 mM NaCl, 1% Nonidet P-40 (NP-40) and 2 mM EDTA. Protein concentrations from each sample were determined by Bradford assay (5000201, BioRad) and 2.5% of the protein lysate was saved as input. Protein A/G coupled Sepharose beads (ab193262, Abcam) were washed with lysis buffer, then equal amounts of protein were incubated with protein A/G beads and either anti-IgG (ab124055, Abcam), anti-RIPK3 (ab62344, Abcam) or anti-MLKL (ab243142, Abcam) primary antibodies overnight at 4 °C with shaking. Protein complexes were collected by centrifugation at 8000×g for 3 min at 4 °C, washed, and subjected to immunoblot analysis.
5
Isolation and Analysis of lncRNA UCA1
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TRIzol reagent (Thermo Fisher Scientific) was used to extract total RNA from PSC cells. mRNA in the total RNA was isolated and purified by PolyATtract® mRNA Isolation Systems (A-Z5300, A&D Technology Corporation, Beijing, China). Anti-EZH2 antibody (36-6300, Thermo Fisher Scientific) or anti-IgG antibody (ab124055, Abcam) was added into IP buffer (20-mM Tris pH 7.5, 140-mM NaCl, 1% NP-40, 2-mM EDTA) to incubate with protein A/g magnetic beads for 1 h for binding. The purified mRNA and magnetic bead-antibody complex were added into IP buffer supplemented with ribonuclease inhibitor and protease inhibitor, and were kept overnight at 4°C. The RNA was eluted with eluent buffer. After extraction and purification by phenol chloroform, lncRNA UCA1 was analyzed by qRT-PCR.
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