The heart grafts were lysed in ice-cold tissue lysis buffer, containing 0.25% NP-40, 125 mM KCl, 10 mM MgCl2, 60 mM HEPES (pH 7.9), 0.5 mM DTT, 0.5mM phenylmethylsulfonyl fluoride, 10 μg/l aprotinin and 10 μg/l leupeptin (all Sangon Biotech Co., Ltd., Shanghai, China). The cell debris was removed by centrifugation and the protein-containing supernatant was removed and titrated according to the specifications of the DC™ Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and stored at −80°C.
Np 40
Manufactured by Sangon
Sourced in China
NP-40 is a non-ionic detergent commonly used in biochemistry and molecular biology applications. It is a mild detergent that helps solubilize and extract proteins from cell membranes while preserving their native structure and function. NP-40 is often used in cell lysis, immunoprecipitation, and Western blotting procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Aprotinin, by Sangon (1 mentions)
Leupeptin, by Sangon (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Triton x 100, by Sangon (1 mentions)
Dithiothreitol (dtt), by Sangon (1 mentions)
Accuri c6, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Sangon (1 mentions)
Sodium mevalonate, by Merck Group (1 mentions)
Puromycin, by Biosharp (1 mentions)
Mg132, by R&D Systems (1 mentions)
Anti flag m2 agarose bead, by Merck Group (1 mentions)
Gw3965, by Cayman Chemical (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Merck Group (1 mentions)
Sumo1 amc, by R&D Systems (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
6 protocols using np 40
1
Heart Tissue Protein Extraction
2
Chromosome Enumeration by FISH
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Putative 1N populations were fixed with Carnoy’s fixative (1:3 acetic acid: methanol) on APES treated slides for 5 min and then washed with 2×SSC (sodium saline citrate buffer) for 10 min at room temperature. The slides were further incubated in 1% paraformaldehyde (Amersco, Solon, USA) for 10 min and 0.1% NP-40 (Sangon, Shanghai, China) in 2×SSC for 10 min, following by a dehydrated procedure in 80%, 90%, and 100% ethanol series for 2 min each. FISH probes (Chr 16, Chr 18, Abbott Molecular, Illinois, USA) were denatured on slides at 78 °C for 8 min on a hot plate and further hybridized at 37 °C in a moist chamber for 48 h. Finally, the slides were mounted with Vecatshield mouting media (Vector, Burlingame, USA) containing Hoechst 33342 (Beyotime, Haimen, China) for nuclei staining as described above.
3
Cas13a Protein Expression and Purification
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The open reading frame (ORF) of Cas13a was synthesized after codon optimization. The Cas13a ORF was then cloned into expression vector Pc013 and transfected into E. coli BL21, which were first grown at 37°C and incubated with IPTG at 16°C. Proteins were purified from lysed bacteria using the Ni-NTA protocol. Aliquots of purified protein were stored at −80°C. Other reagents were purchased from Sangon Co., Ltd. (Shanghai, China), including DTT (A100281), EDTA (A100105), TritonX-100 (A110694), NP-40 (A100109), Chelex-100 (C7901) etc.
4
Evaluation of Apoptosis in Irradiated Cells
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Cells were seeded into six-well plates at a density of 10 6 cells per well. 24 h later, the cells were irradiated with various doses of X-rays (0, 4 or 8 Gy) with or without concurrent parecoxib treatment at a concentration equal to the IC 50 . The cells were incubated for an additional 48 h in medium containing either parecoxib or vehicle, and then harvested (retaining all floating cells), fixed in 75% ethanol at 4°C overnight, and incubated with 50 μg/mL propidium iodide (Sigma), 0.3% NP-40 (Sangon Biotech) and 100 μg/mL RNase A for 30 min at room temperature (Sangon Biotech). The number of cells that underwent apoptosis (sub-G1) was evaluated using BD Accuri C6 (Becton Dickinson). All experiments were repeated at least three times.
5
Characterization of Lipid Metabolism Regulators
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Lovastatin (purity ≥ 98.5%, HPLC) was from Shanghai Pharm Valley. Sodium mevalonate (#4667), anti-FLAG M2 agarose beads (#A2220), phenylmethanesulfonyl fluoride (PMSF, #P7626), protease inhibitor cocktail (#P8340), and β-mercaptoethanol (#M3148) were from Sigma-Aldrich. DiI-LDL was from Yeasen (#20614ES76). GW3965 (#10054) was from Cayman. Lipofectamine RNAiMAX (#13778150) was from ThermoFisher. SUMO1-AMC (#UL-704), ubiquitin-AFC (#U-551-050), and MG132 (#I-130) were from Boston Biochem. Puromycin (#BS111) was from Biosharp. G418 (#345810), pepstatin A (#516481), and ALLN (N-acetyl-leu-leu-norleucinal, #208719) were from Calbiochem. Ni-NTA Agarose (#30230) was from Qiagen. Linear polyethylenimine (#23966-1) was from Polysciences. FuGENE HD (#E2311) and M-MLV RTase (#M1701) were from Promega. Leupeptin (#11034626001) was from Roche. DL-Dithiothreitol (DTT, #A100281) and NP-40 (A100109) were from Sangon Biotech. Lipoprotein-deficient serum (density >1.215 g/ml) and delipidated-fetal calf serum were prepared in our laboratory as described previously (50 (link), 51 (link)). The purified PCSK9 protein was kindly provided by Dr Yan Wang (Wuhan University).
6
Ubiquitylation Analysis Protocol for p62
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For p62 ubiquitylation analysis, cells were lysed in urea buffer (50 mM Tris–HCl pH 8.0, 8 M urea, 40 mM imidazole, 100 mM NaH2PO4 and 0.5% CHAPS) added with protease inhibitor cocktail (Roche, 4693159001). Otherwise, cells were lysed in NP-40 buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40 [Sangon Biotech, A100109], 10% glycerol, 2 mM EDTA, 1 mM DTT) supplemented with protease inhibitor cocktail, then the cell lysate was mixed with antibodies at 4 °C for overnight followed by the addition of protein A/G sepharose beads (Thermo Scientific, 20421). Immunocomplexes were washed and subjected to western blot.
For western blot, cells were harvested and lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA) added with protease inhibitor cocktail. Then resolved cell proteins were separated on SDS polyacrylamide gels, and transferred to polyvinylidene difluoride membrane (PALL, bsp0161). After blocking with 5% (w/v) bovine serum albumin (Sigma-Aldrich, B2064), the membrane was stained with the corresponding primary and secondary antibodies. The specific bands were analyzed with an Odyssey® infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA) and quantified using Image J. All uncropped blots/gels are shown in Supplementary Figs.6 –12 .
For western blot, cells were harvested and lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA) added with protease inhibitor cocktail. Then resolved cell proteins were separated on SDS polyacrylamide gels, and transferred to polyvinylidene difluoride membrane (PALL, bsp0161). After blocking with 5% (w/v) bovine serum albumin (Sigma-Aldrich, B2064), the membrane was stained with the corresponding primary and secondary antibodies. The specific bands were analyzed with an Odyssey® infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA) and quantified using Image J. All uncropped blots/gels are shown in Supplementary Figs.
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