Primary antibodies against β-actin, FAK (Focal adhesion kinase), p-397Tyr-FAK, IKKα (I-kappaB kinase α), IKKβ (I-kappaB kinase β), β-catenin, total-p53, p21, and MnSOD were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). Primary antibodies against UCP2, NF-κB p65, p-536Ser-NF-κB p65, S6 ribosomal protein and cleaved caspase 3, were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against wild type p53 were purchased from MilliporeSigma (Burlington, MA, USA). On a 10% SDS–PAGE gel, 20 μg total protein was electrophoresed, transferred onto a polyvinylidene fluoride membranes, blocked, incubated with a primary antibody and then with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactive bands were visualized using a chemiluminescence solution (Genesee Scientific, El Cajon, CA, USA). Experiments were repeated three times. β-actin was employed as an endogenous control. The pixel densities of the protein bands were quantified using ImageJ.
Mnsod
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Switzerland
MnSOD is a laboratory assay kit that measures the activity of the enzyme manganese superoxide dismutase (MnSOD). MnSOD is a mitochondrial enzyme responsible for the dismutation of superoxide radicals into oxygen and hydrogen peroxide. The MnSOD assay kit provides a quantitative assessment of MnSOD activity in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
P akt, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Nf κb p65, by Santa Cruz Biotechnology (2 mentions)
C jun, by Santa Cruz Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Occludin, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Amaxa nucleofector technology, by Lonza (2 mentions)
Scrambled sirna, by Santa Cruz Biotechnology (2 mentions)
Flag tag (flag), by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Tom20, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Santa Cruz Biotechnology (2 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Cox 2, by Santa Cruz Biotechnology (2 mentions)
29 protocols using mnsod
1
Western Blot Analysis of Cellular Proteins
2
Protein Expression Analysis in TH1 Cells
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The total cellular protein of TH1 cells was extracted using RIPA lysis buffer (Thermo Fisher Scientific). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene fluoride membranes (Sigma-Aldrich). Membranes were blocked with 5% skim milk and incubated with primary antibodies against manganese-dependent superoxide dismutase (MnSOD; Santa Cruz Biotechnology, Dallas, TX, USA), p-Smad2 (Novus Biological, Centennial, CO, USA), Smad7 (R&D system, Minneapolis, MN, USA), fibronectin (Thermo Fisher Scientific), p-Akt (Santa Cruz Biotechnology), PrPC (Santa Cruz Biotechnology), Collagen I (Santa Cruz Biotechnology), E-cadherin (Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA; Santa Cruz Biotechnology), and β-actin (Santa Cruz Biotechnology). After incubation of membranes with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific) in a dark room. For quantification of band intensity, each band intensity was analyzed by ImageJ software (http://rsb.info.nih.gov/ij/ ). The expression levels of proteins were determined relative to the expression of β-actin.
3
Investigating Antioxidant Defenses in UV-Irradiated HaCaT Cells
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HaCaT cells were pretreated with 200 μg/mL RBE and exposed to 100 mJ/cm2 UVB radiation. The protein derived from the treatment was isolated using 1x cell lysis buffer (Cell Signaling), and the concentration was measured using the Bradford Protein Assay Kit (AMRESCO). Protein lysates were evaluated with Western blot analyses as previously described [20 (link), 21 (link)]. Western blot analysis was performed using the specific antibodies: PARP, caspase-3 (DAKO), catalase (Bioss), Cu/ZnSOD (ABBIOTEC), GAPDH, MnSOD, Nrf2, HO-1, β-actin, phos-p38, p38, c-Jun, NFκBp65, and NFκBp50 (Santa Cruz). The levels of GAPDH or β-actin were used as the internal loading control. Densitometric analyses of scanned images were performed using GeneTools software (Syngene, UK).
4
Signaling Pathway Profiling in Cell Culture
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Procaine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor® 488 donkey anti-mouse IgG (H+L) antibody and Fluor® 594 donkey anti-rabbit IgG (H+L) antibody were obtained from Life Technologies (Grand Island, NY, USA). iN-fect™ in vitro Transfection Reagent was obtained from iNtRON Biotechnology (Seongnam, Korea). Antibodies against MnSOD, Fibronectin, Vimentin, E-cadherin, N-cadherin, Occludin, Twist, MMP-2, MMP-9, Akt, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Snail, p-c-Met(Tyr1234/1236), c-Met, p-PI3K(Tyr458), PI3K, p-Akt(Ser473), p-mTOR(Ser2448), mTOR, p-MEK(Ser217/221), MEK, p-ERK(Thr202/Tyr204), and ERK were procured from Cell Signaling Technology (Beverly, MA, USA).
5
Ischemic Thigh Tissue Analysis
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After 1, 3, and 28 days following operation, the ischemic thigh tissues were removed and fixed with 4% paraformaldehyde (Sigma). Each tissue sample was embedded in paraffin. Immunofluorescence staining was performed using primary antibodies against human nuclear antigen (HNA; Millipore, Billerica, MA, USA), manganese-dependent superoxide dismutase (MnSOD; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (Santa Cruz Biotechnology), CD31 (Santa Cruz Biotechnology), and α-SMA (Santa Cruz Biotechnology) and secondary antibodies Alexa-488 and Alexa-594 (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 4′,6-diaminido-2-phenylindol (DAPI; Sigma), and immunostained samples were observed using confocal microscopy (Olympus, Tokyo, Japan).
6
Western Blot Analysis of ER Stress Markers
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The MSCs were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain total cellular protein. Cell lysates (20 µg of total protein) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to the nitrocellulose membrane. After the blots had been washed with TBST (10 mM Tris-HCl (pH 7.60), 150 mM NaCl, 0.05% Tween-20), they were blocked with 5% skimmed milk for 1 h and incubated with appropriate primary antibodies at the dilutions recommended by the supplier. Antibodies against GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, IRE1α, CHOP, p-IRE1α, NF-κB, p-NF-κB, JNK, p-JNK, p38, p-p38, BCL-2, BAX, cleaved caspase-3, cleaved PARP-1, PrPC, MnSOD, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The membranes were then washed, and primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
7
Modulating Cellular Metabolism via siRNA
8
Western Blot Analysis of Oxidative Stress Markers
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Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes, and the membranes were blocked using 5% skim milk in Tris-buffered saline with Tween-20 (TBST). After 1 h, the membranes were incubated with primary antibodies against MMP-8 (1:1000, Abcam), the phospho- or total form of MAP kinases (1:1000, Cell Signaling), HO-1, NQO1, MnSOD, catalase, Nrf2, c-Jun, and lamin A (1:1000, Santa Cruz) at 4 °C overnight. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution in TBST; Bio-Rad Laboratories), and an enhanced chemiluminescence detection kit was used for evaluations (Thermo Fisher Scientific, Waltham, MA).
9
Protein Expression Analysis in Metabolic Regulation
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The following primary antibodies were used in this study: O-GlcNAc (Cat. No. cs9875, Cell Signaling, Danvers, MA), FLAG (Cat. No. ab21536, Abcam), Akt (Cat. No. cs9272, Cell Signaling, Danvers, MA), pSer437-Akt (Cat. No. 9271, Cell Signaling), FOXO1 (Cat. No. ab179450, Abcam), pT24-FOXO1 (Cat. No. ab58517, Cell Signaling), PEPCK (Cat. No. sc32879, Santa Cruz Biotechnology, Dallas, TX), MnSOD (Cat. No. 06–984, Millipore, Billerica, MA), FABP1 (Cat. No. ab7847, Abcam), β-actin (Cat. No. cs4970, Cell Signaling).
10
Western Blot Analysis of MNSOD and Tubulin
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Samples were separated on SDS-PAGE gel. After electrophoresis, the proteins were transferred to nitrocellulose (NC) membranes, blocked then probed with primary antibodies against MNSOD (1:750, Santa Cruz, Dallas, TX, USA) and Tubulin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). Proteins were detected using fluorescence conjugated secondary antibody. The membranes were scanned with Odyssey infrared imaging system (Li-Cor, Lincoln, NE, USA).
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