Tnf α

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, China, Germany, Canada

TNF-α is a cytokine that plays a key role in immune system regulation and inflammatory response. It is a protein that can be measured and quantified using various laboratory techniques.

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Close spelling variants of this product

Anti-mouse TNF-α antibody TNF-α (sc-52746) TNF-α (sc1351) Anti-TNF-α (sc-52746, Rabbit anti-TNF-α TNF-α antibody (sc-52746), Goat anti-TNF-α TNF-α primary antibody Mouse anti-TNF-α Antibodies against TNF-α

209 protocols using tnf α

Western Blot Analysis of Immune Regulators

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Cell lysates were electrophoresed through polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated with polyclonal antibodies specific for NF-κB p65 (Cat. ab72555; abcam; UK), TGFß1 (cat. sc-146; Santa Cruz Biotechnology, Inc; Germany), IRF6 (cat. sc-98829; Santa Cruz Biotechnology, Inc; Germany), TNF-α (cat. LS-C43037; LSBio; Germany) and ß-actin (cat. sc-47778; Santa Cruz Biotechnology, Inc; Germany), and then revealed with secondary antibody. As a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit (cat. Sc-2004; Santa Cruz Biotechnology, Inc; Germany) was used for the primary antibody of NF-κB p65, IRF-6, and TNF-α; whereas, peroxidase-conjugated goat anti-mouse IgG (cat. Sc-2005; Santa Cruz Biotechnology, Inc; Germany) was used for ß-actin primary antibody. Mouse polyclonal anti-ß-actin antibody was used to correct minor differences in protein loading. Finally, the specific signals were detected by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (cat. 34077, Thermo Scientific, Germany). Images were acquired by Quantity One 1-D analysis software (Bio-Rad, Germany).

Qu X., Pröll M., Neuhoff C., Zhang R., Cinar M.U., Hossain M.M., Tesfaye D., Große-Brinkhaus C., Salilew-Wondim D., Tholen E., Looft C., Hölker M., Schellander K, & Uddin M.J. (2015). Sulforaphane Epigenetically Regulates Innate Immune Responses of Porcine Monocyte-Derived Dendritic Cells Induced with Lipopolysaccharide. PLoS ONE, 10(3), e0121574.

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Histological Assessment of GVHD Tissue Damage

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Formalin-fixed, paraffin-embedded tissue sections were stained with H&E, and examined by a pathologist using a semiquantitative scoring system for abnormalities known to be associated with GVHD. The scoring system for each parameter that evaluated both the extent and severity of tissue damage denoted 0 as normal, 0.5 as focal and rare, 1 as focal and mild, 2 as diffuse and mild, 3 as diffuse and moderate, and 4 as diffuse and severe (Cooke et al., 1998 (link)). For TNF-α staining, sections were rehydrated, endogenous peroxidase was eliminated, incubated with TNF-α (Santa Cruz, USA) antibody followed by HRP conjugated secondary antibody and developed with DAB system.

Lim J.Y., Ryu D.B., Lee S.E., Park G., Choi E.Y, & Min C.K. (2015). Differential Effect of MyD88 Signal in Donor T Cells on Graft-versus-Leukemia Effect and Graft-versus-Host Disease after Experimental Allogeneic Stem Cell Transplantation. Molecules and Cells, 38(11), 966-974.

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Intestinal Inflammatory Protein Analysis

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Immunoblotting was performed as described previously [16 (link)]. Briefly, dissected intestinal were homogenized and lysed with lysis buffer. Protein extract was separated on SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane and probed with the following antibodies: IL-6, IL-1β, TNF-α, PGE2, COX-2, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA). Band intensity was determined by densitometry analysis using the ImageJ analysis system.

Park S.A., Lee G.H., Hoang T.H., Lee H.Y., Kang I.Y., Chung M.J., Jin J.S, & Chae H.J. (2021). Heat-inactivated Lactobacillus plantarum nF1 promotes intestinal health in Loperamide-induced constipation rats. PLoS ONE, 16(4), e0250354.

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Vesicle Uptake in Activated HUVECs

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HUVECs (6-well plates) were treated with 50 ng/ml TNF-α (carrier free, Santa Cruz, CA) for 4 h. The activated HUVECs were then harvested and incubated with 2 and 20 μg/ml vesicles, pre-stained by lipophilic tracer benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-, perchlorate (DiO, Cat. No. V22889, Invitrogen, Waltham, MA), with or without additional cholesterol) at 37 °C for 30 min. The cells were then analyzed for fluorescent intensity by Gallios flow cytometer (Beckman coulter, Brea, CA).

Gao J., Su Y, & Wang Z. (2023). Remote Co-loading of amphipathic acid drugs in neutrophil nanovesicles infilled with cholesterol mitigates lung bacterial infection and inflammation. Biomaterials, 296, 122071.

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BDNF Signaling Protein Detection

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BDNF, leupeptin, aprotinin, phenyl-methylsulfonyl fluoride, pepstatin A, soybean trypsin inhibitor, NaF, sodium vanadate, glycerophosphate, 2-mercaptoethanol, NMDA, glycine, Tween 20, NP-40, and Histopaque-1077 were from Sigma. Anti-TrkB (SC-8316), -pY-Trk (SC-8058), -phospholipase C-γ1 (SC-7290), -N-Shc (SC-365598), -NR1 (SC-9058), -TNFα (SC-52746), -IL-6 (SC-57315), -IL-1β (SC-57315), -Arc (SC-365736), - BDNF/proBDNF (SC-2098), –NT3 (SC-547), -NT4 (SC-545), -β-actin (SC-47778) were from Santa Cruz Biotechnology. FNDC-5/irisin (36–335) was from ProSci (Poway, CA, USA). Seize-X immunoprecipitation kit, antigen elution buffer, Bind NeutrAvidin, high binding capacity coated 96-well plates, and West Pico chemiluminescent reagents were from Pierce-Endogen (Rockford, IL, USA). Bradford reagent, SDS-PAGE reagents, and pre-stained molecular weight markers were from Bio-Rad. Protease inhibitors (EDTA-free) and protein phosphatase inhibitor tablets were from Roche (Basel, Switzerland). BDNF was reconstituted according to the manufacturer’s instruction. To avoid freezing damage, 10% glycerol was added for a final concentration of 10 ng/μl BDNF and stored in 80°C until use. All other test agents were made fresh according to the manufacturer’s recommendation. The DMSO concentration in the incubation medium was 1%.

Meade G.M., Charron L.S., Kilburn L.W., Pei Z., Wang H.Y, & Robinson S. (2020). A Model of Negative Emotional Contagion Between Male-Female Rat Dyads: Effects of Voluntary Exercise on Stress-Induced Behavior and BDNF-TrkB Signaling. Physiology & behavior, 234, 113286.

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Hippocampal Protein Profiling by Western Blot

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Hippocampal slices were homogenized with Tris-HCl buffer including (in mM) 0.5 EDTA and 250 sucrose and centrifuged at 15,000 rpm for 25 min at 4°C. The protein was extracted from supernatant by SDS-PAGE on 10% acrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% nonfat dry milk in a Tris-Buffered Saline Tween-20 (TBST) buffer (50 Tris-HCl, 150 mmol/l NaCl, 0.1% Tween, pH 7.4) for 1 h at RT. For primary antibodies, p-PKC was purchased from Abcam (Cambridge, UK), p-ERK and TNF-α from Santa Cruz (CA, USA). The peroxidase-labeled affinity purified secondary antibody rabbit IgG and mouse IgG were purchased from Sigma (St. Louis, USA). After overnight incubation of primary antibody followed by 1-hour incubation of secondary antibody, blots were detected by X-ray film and quantified densitometric measurements through three independent repeats.

Zheng X., Zhou J, & Xia Y. (2015). The role of TNF-α in regulating ketamine-induced hippocampal neurotoxicity. Archives of Medical Science : AMS, 11(6), 1296-1302.

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Retinal Protein Expression Analysis

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Six eyeballs were enucleated, and retinal tissues were collected and placed into Eppendorf tubes with 200 μL of lysate. The remaining tissues- “eyecups” were also mounted in tissue culture plate (Corning Incorporated, Corning, NY, USA) and up to 5 μL of lysate was added into the eyecups to extract retinal pigment epithelial proteins. After 5 min, the lysates were collected. Subsequent steps were performed in accordance with a conventional protocol. Equal amounts of protein samples were used for SDS-PAGE electrophoresis and transmembrane incubation with GLUT-1 (Millipore Corporation. St. Charles, MI, USA), ICAM-1, TNF-α (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and albumin (Abcam plc, Cambridge, UK) primary antibodies. The following day, secondary antibody incubation was conducted at room temperature for 1 h after the membranes were washed three times. Finally, the relative densities of the blots were measured by UVP GelDoc-It Imager (UVP LLC, Upland, CA, USA).

You Z.P., Zhang Y.L., Shi K., Shi L., Zhang Y.Z., Zhou Y, & Wang C.Y. (2017). Suppression of diabetic retinopathy with GLUT1 siRNA. Scientific Reports, 7, 7437.

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Histological Analysis of Cardiac Fibrosis

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Hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin (H&E) (C0105, Beyotime Biotechnology) for histological analysis and Sirius Red and Masson’s trichrome (G1340-7 × 100 ml, Solarbio Life Science) to evaluate cardiac fibrosis. The sections were observed under a light microscope (Nikon, Japan).
For immunohistochemical staining, the sections were deparaffinized and rehydrated. The sections were treated with 3% H2O2 for 30 min to block endogenous peroxidase activity and then with 1% BSA in PBS for 30 min. The slides were incubated overnight at 4°C with the primary antibody (TNF-α, 1:50; both from Santa Cruz). Peroxidase-conjugated secondary antibodies were used for detection (Santa Cruz; 1:100 dilution; 1 h incubation). The slides were counterstained with hematoxylin for 5 min, dehydrated, and mounted. Images were viewed by a bright field microscope (Nikon).

Ye S., Su L., Shan P., Ye B., Wu S., Liang G, & Huang W. (2021). LCZ696 Attenuated Doxorubicin-Induced Chronic Cardiomyopathy Through the TLR2-MyD88 Complex Formation. Frontiers in Cell and Developmental Biology, 9, 654051.

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Molecular Mechanisms of Cell Injury

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Antibodies RIP1, RIP3, KIM-1, Nox2, Nox4, TNF-α, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Rabbit anti-P-MLKL and Anti-cleaved-caspase-3, cleaved-caspase-12 were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Lipofectamine 3000 was purchased from Science Biotechnology (Invitrogen, Beijing, China). Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Cell Malondialdehyde (MDA) assay kit (Colorimetric method), reduced glutathione (GSH) assay kit, creatinine (Cr) Assay kit (sarcosine oxidase) and urea (BUN) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China). Reactive Oxygen Species Assay (DCF Assay) Kit and Dihydroethidium (DHE) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). ITC AnnexinV/propidiuiodide was purchased from Bestbio (Shanghai, China). Mouse NGAL ELISA kit was purchased from CUSABIO (Wuhan, China).

Gao L., Wu W.F., Dong L., Ren G.L., Li H.D., Yang Q., Li X.F., Xu T., Li Z., Wu B.M., Ma T.T., Huang C., Huang Y., Zhang L., Lv X., Li J, & Meng X.M. (2016). Protocatechuic Aldehyde Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Nox-Mediated Oxidative Stress and Renal Inflammation. Frontiers in Pharmacology, 7, 479.

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Garcinol and PTZ Neurochemical Interactions

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Garcinol and pentylenetetrazole (PTZ) were procured from Sigma-Aldrich (St. Louis, MO, USA). PTZ was dissolved in freshly prepared saline prior to the injection. Antibodies against BDNF, TrkB, GABA_A, and GAD65 (Cell Signalling Technology, Danvers, MA, USA), caspase-3, Bcl-2, Bad, Bax, Bcl-xL, and TNF-α ((Santa Cruz Biotechnology, Santa Cruz, CA,USA) were used for expression analysis. All other standard chemicals used in this study were of analytical grade procured from Sigma-Aldrich (St. Louis, MO, USA).

Hao F., Jia L.H., Li X.W., Zhang Y.R, & Liu X.W. (2016). Garcinol Upregulates GABAA and GAD65 Expression, Modulates BDNF-TrkB Pathway to Reduce Seizures in Pentylenetetrazole (PTZ)-Induced Epilepsy. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 4415-4425.

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