Radioimmunoprecipitation assay ripa lysis buffer

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The Radioimmunoprecipitation Assay (RIPA) Lysis Buffer is a complex solution used for the extraction and solubilization of proteins from biological samples. It is designed to disrupt cell membranes and facilitate the release of intracellular proteins while maintaining their native structure and function.

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RIPA lysis buffer (432 citations) RIPA buffer (365 citations) RIPA (33 citations) Radioimmunoprecipitation assay buffer (31 citations) Lysis buffer (9 citations) Radioimmunoprecipitation lysis buffer (4 citations) RIPA) assay buffer (3 citations) Radioimmunoprecipitation (RIPA) buffer (2 citations)

8 protocols using radioimmunoprecipitation assay ripa lysis buffer

Apoptosis Signaling Pathway Activation

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CTAB was purchased from Sigma-Aldrich; Merck KGaA. PI3K agonist 740 Y-P were purchased from MedChemExpress (MCE). Antibodies against caspase-3 (1:1,000 dilution; cat. no. 9662), cleaved caspase-3 (1:1,000 dilution; cat. no. 9661), caspase-8 (1:1,000 dilution; cat. no. 9746), cleaved caspase-8 (1:1,000 dilution; cat. no. 9496), caspase-9 (1:1,000 dilution; cat. no. 9502), cleaved caspase-9 (1:1,000 dilution; cat. no. 20750), PARP (1:1,000 dilution; cat. no. 9532), cleaved PARP (1:1,000 dilution; cat. no. 32563), AKT (1:1,000 dilution; cat. no. 9272) and p-AKT (1:1,000 dilution; cat. no. 9271) were purchased from Cell Signaling Technology. Antibodies against PI3K (1:1,000 dilution; cat. no. 180967), p-PI3K (1:1,000 dilution; cat. no. 278545), Bcl-2 (1:1,000 dilution; cat. no. 32124), Bax (1:1,000 dilution; cat. no. 32503), cytochrome c (1:5,000 dilution; cat. no. 133504), and β-actin (1:1,000 dilution; cat. no. 8226) and secondary antibodies (1:5,000 dilution; cat. no. 96899 and 96879) were obtained from Abcam. Phosphate-buffered saline (PBS) was obtained from Gibco; Thermo Fisher Scientific, Inc. Radioimmunoprecipitation assay (RIPA) lysis buffer was purchased from Santa Cruz Biotechnology.

Da W., Tao L, & Zhu Y. (2021). The inhibitory effect of CTAB on human osteosarcoma through the PI3K/AKT signaling pathway. International Journal of Oncology, 59(1), 42.

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Immunoblotting for Insulin Signaling

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HOCl and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Sigma-Aldrich (St. Louis, MO). Protein A/G-agarose, radioimmunoprecipitation assay (RIPA) lysis buffer, and antibodies against β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-IR-β (Tyr1150/1151), IR-β, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, and horseradish peroxidase–linked secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibody against MPO was from R&D Systems (Minneapolis, MN). Antibodies against nitrotyrosine (NT), uncoupling proteins 1 (UCP1), and UCP3 were from Millipore Corp. (Billerica, MA). The enhanced chemiluminescence detection kit was obtained from Pierce (Rockford, IL).

Wang Q., Xie Z., Zhang W., Zhou J., Wu Y., Zhang M., Zhu H, & Zou M.H. (2014). Myeloperoxidase Deletion Prevents High-Fat Diet–Induced Obesity and Insulin Resistance. Diabetes, 63(12), 4172-4185.

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Furanodiene Anti-Cancer Mechanism

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Furanodiene (FUR) was purchased from the National Institutes for Food and Drug Control. The Roswell Park Memorial Institute-1640 (RPMI-1640) was used as the culture medium and was purchased from Gibco (Gaithersburg, MD, United States). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (PS), and 0.25% (w/v) trypsin/1 mM EDTA were obtained from Invitrogen (Carlsbad, CA, United States). 3-[4, 5-Dimethyl-2-thiazolyl]-2, 5-diphenyltetrazolium bromide (MTT), 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), CellROX^®Deep probe, propidium iodide (PI), and Annexin V/PI detection kit were obtained from Molecular Probes (Eugene, OR, United States). Doxorubicin (DOX) and tert-Butyl hydroperoxide (TBHP) solution were supplied by Sigma-Aldrich (St. Louis, MO, United States). TNF-α immunoassay kit was purchased from R&D Systems (Minneapolis, MN, United States). Radioimmunoprecipitation assay (RIPA) lysis buffer and primary antibodies against TNF-R1 and p65 were obtained from Santa Cruz (Santa Cruz, CA, United States). Primary antibodies against p-IKKα/β (Ser176/180), IKKα, IKKβ, Bcl-xL, Bax, Bad, Caspase-7, Caspase-8, PARP, GAPDH, and β-actin, as well as the secondary antibodies were purchased from Cell Signaling (Danvers, MA, United States). siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).

Zhong Z.F., Yu H.B., Wang C.M., Qiang W.A., Wang S.P., Zhang J.M., Yu H., Cui L., Wu T., Li D.Q, & Wang Y.T. (2017). Furanodiene Induces Extrinsic and Intrinsic Apoptosis in Doxorubicin-Resistant MCF-7 Breast Cancer Cells via NF-κB-Independent Mechanism. Frontiers in Pharmacology, 8, 648.

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Protein Expression Analysis by Western Blot

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Protein extracts were prepared by lysing cells in Radio-Immunoprecipitation Assay (RIPA) lysis buffer from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cleared lysates were separated on 10% Tris-glycine gels followed by transfer onto nitrocellulose membranes. After blocking in 5% skim milk, blots were probed using primary antibodies including β-actin antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA), HuR antibody (1:500, Millipore, Temecula, CA, USA), MMP2 antibody (1:500, Abcam, Cambridge, MA, USA), MMP9 antibody (1:500, Abcam, Cambridge, MA, USA), nuclear factor κB antibody (NF-κB p65, 1:1000, Cell Signaling Technology, Danvers, MA, USA) and phospho-p65 Ser536 (1:1000, Cell Signaling Technology, Danvers, MA, USA). After a wash and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, protein bands were visualized by use of the chemiluminescent HRP substrate (Millipore, Temecula, CA, USA). The intensities of individual bands were measured by densitometry (Model GS-700, Imaging Densitometer; Bio-Rad, Hercules, CA, USA). All experiments were repeated for four times and the mean values derived.

Kong J., Zhang Y., Liu S., Li H., Liu S., Wang J., Qin X., Jiang X., Yang J., Zhang C, & Zhang W. (2017). Melatonin attenuates angiotensin II-induced abdominal aortic aneurysm through the down-regulation of matrix metalloproteinases. Oncotarget, 8(9), 14283-14293.

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Apoptosis Pathway Regulation by ICTS

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ICTS (>98%) was purchased from ChemFaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO) to produce a stock solution. A working solution was diluted from the stock solution using cell culture medium. Hoechst 33342 stain, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), propidium iodide (PI), 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC-1), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Sigma-Aldrich (St. Louis, USA). Crystal violet staining solution and DNA loading buffer were obtained from Beyotime Inc. (Haimen, China). Specific antibodies against Bcl-2, bcl-X protein (Bcl-XL), bcl-2-associated X protein (BAX), bcl-2 homologous antagonist-killer protein (BAK), poly-ADP-ribose polymerase (PARP), caspase-3, caspase-9, phospho-c-Jun N-terminal protein kinase (p-JNK), phospho-extracellular regulated protein kinase 1/2 (p-ERK), phospho-p38 mitogen-activated protein kinase (p-p38), JNK, p38, ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and related secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA). Radio immunoprecipitation assay (RIPA) lysis buffer was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, USA).

Zhang X., Luo W., Zhao W., Lu J, & Chen X. (2015). Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells. Journal of Breast Cancer, 18(2), 112-118.

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Molecular Profiling Employing Mouse Model

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C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Primary antibodies, AhR, CRM1 (A-7), CYP1A1(H-70), E-cadherin (H-108), p53 (Pab 1801), and SOD1 (11407); biotinylated secondary antibody; and Radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies including, phospho-AKT (S473-D9E), BCL-XL (54H6), Nrf2 (D1Z9C), p21 (12D1), and GAPDH (D16H11-XP™) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). 10× Tris-buffered saline (TBS), 20× Tween, SYBR Green one step kits, and chemiluminescence kits were purchased from BIORAD laboratories (Hercules, CA). Diaminobenzidine tetrahydrochloride (DAB) and avidin-biotin complex (ABC) kits were purchased from vector laboratories (Burlingame, CA). RT-PCR primers listed in Table 1 were purchased from Eurofins Scientific (Bellevue, OH).

Marshall K., Liu Z., Olfert I.M, & Gao W. (2020). Chronic Electronic Cigarette Use Elicits Molecular Changes Related to Pulmonary Pathogenesis. Toxicology and applied pharmacology, 406, 115224.

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Tissue Extraction and Protein Quantification

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CRFOE (n=10; 5 male and 5 female) and control mice (n=10; 5 male and 5 female) used for western blotting and ELISA analyses were briefly exposed to isoflurane and then euthanized by decapitation via guillotine. Brain tissue was rapidly removed on ice from each animal and using a trephine and razor blades and the medial prefrontal cortex and LC brain regions were micro-dissected from each. The brain regions of each animal were homogenized with a pestle and extracted in RadioImmunoPrecipitation Assay (RIPA) lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) on ice for 20 min. Lysates were cleared by centrifugation at 13,000 rpm for 12 min at 4 °C. Protein concentrations of the supernatants were quantified using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL).

Ross J.A., Alexis R., Reyes B.A., Risbrough V, & Van Bockstaele E.J. (2019). Localization of Amyloid Beta Peptides to Locus Coeruleus and Medial Prefrontal Cortex in Corticotropin Releasing Factor Overexpressing Male and Female Mice. Brain structure & function, 224(7), 2385-2405.

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Western Blot Analysis of DNA Damage Proteins

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Protein was extracted with Radioimmunoprecipitation Assay (RIPA) Lysis buffer (Santa Cruz Biotechnology). Protein concentration was determined by the Pierce BCA assay method according to the manufacturer's protocol (Thermo Scientific). Western blots were performed with 10 µg protein per lane using semi-dry transfer and horseradish-peroxidase (HRP)-conjugated antigen retrieval system as described previously (Kleiman et al. 2013) (link). The nitrocellulose membranes were probed with the following primary antibodies: UCHL1 (D3T2E) (#13179), CHK2 (1C12) (#3440), phosphorylated-CHK2 (Thr68) (2661) (Cell Signaling Technology), β-tubulin (ab6046), β-actin (ab8229) and anti-GAPDH (EPR16891) (ab181602) (Abcam).

Finnerty B.M., Moore M.D., Verma A., Aronova A., Huang S., Edwards D.P., Chen Z., Seandel M., Scognamiglio T., Du Y.N., Elemento O., Zarnegar R., Min I.M, & Fahey T.J. (2019). UCHL1 loss alters the cell-cycle in metastatic pancreatic neuroendocrine tumors. Endocrine-related cancer, 26(4).

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