SRA 01/04 cells, treated either with 200 μM H2O2 or 1% ERK inhibitor PD98059, as well as controls, and human anterior capsule membrane samples were homogenized and lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium pyrophosphate, protease inhibitor cocktail). The total protein concentration in the extracts was determined using the BCA reagent (Pierce). Equal amounts of proteins were separated by 15% SDS–PAGE gel, transferred to PVDF membranes, and incubated with appropriate primary and secondary antibodies, following the standard procedures. The primary antibodies were anti-Grx1 (1:1000; 15,804-1-AP, Proteintech), anti-actin (1:60,000; clone AC-74, Sigma), anti-tublin (1:100,000; Calbiochem, Gibbstown, NJ), anti-Bcl (1:500; BD Biosciences, San Diego, CA), anti-Bax (1:5000; Cell Signaling Technology, Danvers, MA), and anti-ERK (1:1000; #4376, Cell Signaling Technology), anti-p-ERK (1:1000; #4370, Cell Signaling Technology), and anti-SOD (1:1000; #2770, Cell Signaling Technology). The secondary antibodies were anti-rabbit or anti-mouse (both from Jackson Laboratories, Bar Harbor, ME). As previously reported [27 (link)], quantitative analysis of the band intensity of the western blots was performed using Image J software (v1.51, National Institutes of Health, Bethesda, MD) to determine changes in the protein levels.
Anti sod
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SOD is a laboratory reagent that detects superoxide dismutase (SOD) proteins. SOD is an essential antioxidant enzyme that catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide. Anti-SOD can be used to measure SOD levels and activity in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Anti apx l, by Cell Signaling Technology (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti cat, by Cell Signaling Technology (1 mentions)
As06 172, by Agrisera (1 mentions)
Hrp linked anti rabbit 1gg, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Sodium thiosulphate, by Merck Group (1 mentions)
Tyrosol, by Merck Group (1 mentions)
Anhydrous sodium carbonate, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Euroclone (1 mentions)
Ethylene glycol bis β aminoethyl ether n n n n tetraacetic acid egta, by Merck Group (1 mentions)
Anti catalase, by Cell Signaling Technology (1 mentions)
2 2 azino bis 3 ethylbenzothiazoline 6 sulphonic acid, by Merck Group (1 mentions)
2 2 4 trimethylpentane, by Merck Group (1 mentions)
5 protocols using anti sod
1
Immunoblot Analysis of Oxidative Stress Markers
2
Western Blotting of Photosynthetic Proteins
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For Western blotting, leaves were homogenized in extraction buffer as described previously [9 (link)]. The protein concentration was determined by the Bradford method using BSA (bovine serum albumin) as a standard curve. After electrophoresis, the gels were transferred to 0.45 μM nitrocellulose membrane (Sigma-Aldrich) and were further processed for Western blots as described in our previous studies [9 (link)]. The following primary antibodies were used: polyclonal primary antibodies 1:1000 dilution of anti PsaA (Agrisera # AS06 172) for PsaA, anti PsbA (Agrisera # AS05 084) for PsbA, anti-SOD (Cell Signaling #2770) for superoxide dismutase, 1:1000 dilution of anti-CAT (Cell Signaling #12980) for catalase, and 1:1000 dilution of anti-APX/L (Cell signaling #AS08 368) for ascorbate peroxidase. The blots were treated with 1:1000 dilution horseradish-linked anti-rabbit 1gG (Cell Signaling #7074) for 1 h as a secondary antibody.
3
Immunoblot Analysis of Antioxidant Enzymes
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For the immunoblot analysis the same protein extraction was followed as for first dimensional gel electrophoresis (protocol given above). After electrophoresis, the gels were transferred to 0.45 µM nitrocellulose membrane (Sigma-Aldrich). After protein transfer, the blots were blocked with 5 % nonfat dry skimmed milk or bovine serum albumin (Sigma-Aldrich). After washing with TBS, the blots were incubated with monoclonal primary antibodies 1:1000 dilution of anti-SOD (Cell Signaling #2770, Danvers, MA, USA) for superoxide dismutase, and 1:1000 dilution of anti-APX/L (Cell signaling #AS08 368) for ascorbate peroxidase overnight at 4 °C. The blots were treated with 1:1000 dilution HRP-linked anti-rabbit 1gG (Cell Signaling #7074) for 1 h as a secondary antibody. Chemiluminescence reactions were performed with super signal west pico chemiluminescent substrates (Cell Signaling SignalFireTM ECL Reagent #6883) and images were captured on ChemiDoc imaging system (BioRad, Hercules, CA, USA).
4
Antioxidant Activity Evaluation Protocol
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Phenolphthalein 1%, Folin–Ciocalteau reagent, and formaldehyde 40% (m/v) were purchased from Titolchimica (Pontecchio Polesine, Italy). Acetic acid, ethanol (99.8%), methanol, sodium carbonate anhydrous, diethyl ether, 2,2,4-trimethylpentane, hexane, hydrochloric acid 37%, sodium hydroxide 0.1 N, sodium thiosulphate 0.01 N, potassium persulfate, potassium iodine, chloroform, starch solution indicator 1%, as well as 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), tyrosol, the standard of Trolox, tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), NaCl, NaF, H2O2, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraAcetic acid (EGTA), and Triton were purchased from Sigma Aldrich (Milan, Italy). Hydroxytyrosol was obtained from Cayman Chemicals, Vinci Biochem (Vinci, Italy). DCFH2-DA (2,7-dichlorodihydrofluorescein diacetate) was purchased from Invitrogen (Milan, Italy). The standard gallic acid was supplied by Carlo Erba (Milan, Italy). Fetal calf serum was from EuroClone SpA (Milan, Italy). Diff-Quik was from Mertz-Dade AG, Dade International, (Milan, Italy), anti-caspase-3, anti-catalase and anti-SOD were from Cell Signaling, (Milan, Italy), secondary antibodies were from Promega (Padova, Italy), and cell culture dish and plates were from Sarstedt (Verona, Italy).
5
RNA and Protein Extraction Methods
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Total RNA was extracted with the help of the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription and qPCR techniques were performed utilizing the PrimeScript™ RT Master Mix and SYBR Premix ExTaq (Takara, Kyoto, Japan), based on the kit instructions. The data were assessed using the 2-ΔΔCt technique.
Total proteins were extracted using the RIPA buffer, then loaded uniformly onto the SDS-PAGE (sodium dodecyl sulfate, 10%) gel, and transferred to the nitrocellulose membranes. The membranes were then blocked with bovine serum albumin (BSA, 5%) and incubated with the primary antibodies such as anti-FTH1 (ABclonal, A19544), anti-SOD (Cell Signaling Technology, 71G8), anti-ATG7 (Abcam, ab52472), and anti-GAPDH (Sigma-Aldrich, USA). Thereafter, the membranes were washed and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Finally, the membranes were washed and imaged using the ChemiDoc XRS+ System (Bio-Rad Laboratories, USA).
Total proteins were extracted using the RIPA buffer, then loaded uniformly onto the SDS-PAGE (sodium dodecyl sulfate, 10%) gel, and transferred to the nitrocellulose membranes. The membranes were then blocked with bovine serum albumin (BSA, 5%) and incubated with the primary antibodies such as anti-FTH1 (ABclonal, A19544), anti-SOD (Cell Signaling Technology, 71G8), anti-ATG7 (Abcam, ab52472), and anti-GAPDH (Sigma-Aldrich, USA). Thereafter, the membranes were washed and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Finally, the membranes were washed and imaged using the ChemiDoc XRS+ System (Bio-Rad Laboratories, USA).
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