Biotinylated 15d pgj2

Manufactured by Cayman Chemical

Sourced in United States

Biotinylated 15d-PGJ2 is a synthetic prostaglandin derivative with a biotin moiety attached. It is a useful tool for research applications.

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15d-PGJ2

5 protocols using biotinylated 15d pgj2

Antibody Preparation and Reagent Sourcing

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Antibody against FLAG was purchased from Sigma-Aldrich, GFP and HA from Santa Cruz Biotechnology. Antibody against eIF4GI was prepared in our laboratory (Kim et al., 2005 (link)). Chemicals 15d-PGJ2, biotinylated 15d-PGJ2 and PGE2 were purchased from Cayman Chemical. Sodium arsenite was purchased from Sigma-Aldrich. Immobilized streptavidin agarose was purchased from Pierce.

Yun S.J., Kim H., Jung S.H., Kim J.H., Ryu J.E., Singh N.J., Jeon J., Han J.K., Kim C.H., Kim S., Jang S.K, & Kim W.J. (2018). The mechanistic insight of a specific interaction between 15d-Prostaglandin-J2 and eIF4A suggests an evolutionary conserved role across species. Biology Open, 7(11), bio035402.

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Evaluating Oxidative Effects on sEH Adduction

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To assess if oxidation affected electrophilic 15d-PGJ₂ adducting to sEH, biotinylated-15d-PGJ₂ (Cayman) was used in an adaptation of the enzymatic assay method described above. Briefly, full-length protein sEH protein was reduced with 10 μM TCEP on ice for 15 min, then diluted in freshly prepared 25 mM TRIS-HCl pH 7.4 to a final concentration of 322 nM. The proteins were then incubated at room temperature for 10 min with or without H₂O₂ (final concentration 1 μM), the proteins were then subsequently incubated at room temperature for 30 min with biotinylated-15d-PGJ₂ (final concentration 10 μM). Protein samples were then re-suspended with an equal volume of 2x reducing Laemmli SDS sample buffer (total volume 100 μL). Protein samples were loaded and separated by SDS-PAGE using Mini-protean 3 system (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). Blots were incubated with the biotin-antibody (#D5A7, Cell Signaling) diluted 1:1000 in PBS-Tween 5% milk overnight. One hr of incubation with horseradish peroxidase-coupled anti-rabbit (#7074, Cell Signaling) IgG secondary antibody (diluted 1:1000 in PBS-Tween in 5% milk) was used to detect the primary antibody, along with ECL reagent (ThermoFisher).

Charles R.L., Abis G., Fernandez B.F., Guttzeit S., Buccafusca R., Conte M.R, & Eaton P. (2021). A thiol redox sensor in soluble epoxide hydrolase enables oxidative activation by intra-protein disulfide bond formation. Redox Biology, 46, 102107.

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Bioactive Lipid Signaling in Cellular Antioxidant Response

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15d-PGJ₂, 9,10-dihydro-15d-PGJ₂, and biotinylated 15d-PGJ₂ were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Biotinylated 9, 10-dihydro-15d-PGJ₂ was kindly provided from Dr. Young-Ger Suh (Cha University, Seongnam, South Korea). RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). Antibodies against NRF2, HO-1, VEGF, and actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). ZnPP was provided from OXIS International Inc. (Portland, OR, USA). The anti-rabbit and anti-mouse horseradish peroxidase conjugated secondary antibodies were purchased from Zymed Laboratories (San Francisco, CA, USA). The enhanced chemiluminescence (ECL) and [γ-³²P] ATP were supplied from Amersham Pharmacia Biotech (Buckinghamxhire, UK). Antioxidant responsive element (ARE) or GC-mutated ARE-driven luciferase porter plasmid and dominant-negative (DN) NRF2 were kindly provided by Prof. Jeffrey A. Johnson (University of Wisconsin-Madison, WI, USA). The following oligonucleotides were used (core sequence is underlined, and mutated bases are in bold): wild-type ARE, 5′-CTC AGC CTT CCA AAT CGC AGT CAC AGT GAC TCA GCA GAA TC-3′; and GC mutant ARE, 5′-CTC AGC CTT CCA AAT CGC AGT CAC AGT GAC TCA ATA GAA TC-3′. siRNAs directed for NRF2 and HO-1 were produced from Dharmacon, Inc. (Lafayette, CO, USA).

Kim E.H., Kim S.J., Na H.K., Han W., Kim N.J., Suh Y.G, & Surh Y.J. (2021). 15-Deoxy-Δ12,14-prostaglandin J2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells. Cells, 10(3), 526.

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NLRP3 Inflammasome Regulation by 15d-PGJ2

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C57BL/6J wild type or NLRP3-deficient BMDMs were stimulated with LPS (1 μg/mL, 4 h) to induce upregulation of NLRP3. Cells were then incubated with 15d-PGJ2 or biotinylated 15d-PGJ2 (Cayman Chemical) (50 μM, 1 h). Sucrose buffer lysates were made in the presence of Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Approximately 200 μg of cell lysate was mixed with 5 μg of anti-NLRP3 antibody (MAB7578, R&D Systems) and placed on a rotary shaker at 4°C for 2 h. Protein A/G agarose slurry (20 μL, Santa Cruz Biotechnology) was added and the incubation was continued overnight. Bound immune complexes were washed with PBS four times prior to elution of proteins in 2X SDS loading buffer. Biotinylated adducts were detected with IR680-conjugated streptavidin (LI-COR Bioscienses) by Western blotting.

Maier N.K., Leppla S.H, & Moayeri M. (2015). The cyclopentenone prostaglandin 15d-PGJ2 inhibits the NLRP1 and NLRP3 inflammasomes. Journal of immunology (Baltimore, Md. : 1950), 194(6), 2776-2785.

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Proteomic Analysis of Lipid Mediators

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Leibovitz's/L15 medium, trypsin, deoxyribonuclease I, fetal bovine serum, horse serum, penicillin, and streptomycin were obtained from Invitrogen. PGD₂, Δ¹²‐PGJ₂, 15d‐PGJ_2, and biotinylated 15d‐PGJ₂ were obtained from Cayman Chemical. 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H tetrazolium bromide (MTT) was purchased from DOJINDO. Immobiline™ DryStrip Gels (pH3‐10) and Amersham ECL Plus™ Western Blotting Detection Reagents were obtained from GE Healthcare Bio‐Sciences Corp. Sequence grade modified trypsin was purchased from Promega, and N‐(1‐pyrenyl) iodoacetamide was from Molecular Probes. Horseradish peroxidase‐linked antibody against biotin was obtained from Cell Signaling Technology. The protein concentration was measured using the BCA protein assay reagent obtained from Thermo Fisher Scientific. 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) was obtained from Pierce. Hoechst 33342 was purchased from Nacalai Tesque. All other chemicals were of reagent grade (Yamamoto et al., 2015b).

Koma H., Yamamoto Y., Okamura N, & Yagami T. (2020). A plausible involvement of plasmalemmal voltage‐dependent anion channel 1 in the neurotoxicity of 15‐deoxy‐Δ12,14‐prostaglandin J2. Brain and Behavior, 10(12), e01866.

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