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Anti 3nt

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Anti-3NT is a laboratory reagent used to detect the presence of 3-nitrotyrosine (3-NT) in biological samples. 3-NT is a biomarker indicative of nitrosative stress and can be used to study various physiological and pathological conditions. Anti-3NT provides a tool for researchers to quantify and analyze 3-NT levels in their investigations.

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Lab products found in correlation

Anti 4 hne, by Alpha Diagnostic (3 mentions) Testicular hyaluronidase, by Merck Group (2 mentions) Goat anti rabbit igg h l biotinylated ba 1000, by Vector Laboratories (2 mentions) Abc hrp kit, by Vector Laboratories (2 mentions) Sk 4100, by Vector Laboratories (2 mentions) Image lab software, by Bio-Rad (2 mentions) Anti histone h3, by Santa Cruz Biotechnology (2 mentions) Anti nrf2, by Santa Cruz Biotechnology (2 mentions) Anti actin, by Santa Cruz Biotechnology (2 mentions) Anti bax, by Cell Signaling Technology (2 mentions) Anti caspase 8, by Cell Signaling Technology (2 mentions) Anti caspase 3, by Cell Signaling Technology (2 mentions) Anti rabbit or mouse igg alkaline phosphatase secondary antibodies, by Merck Group (1 mentions) Chemidoc xp image system, by Bio-Rad (1 mentions) Infinite m200, by Tecan (1 mentions) Anti tgf β1, by Cell Signaling Technology (1 mentions) Anti gapdh, by Santa Cruz Biotechnology (1 mentions) Anti col4, by Abcam (1 mentions) Anti pdcd4, by Santa Cruz Biotechnology (1 mentions) Anti t jnk, by Cell Signaling Technology (1 mentions)

8 protocols using anti 3nt

1

Immunohistochemical Analysis of 3-Nitrotyrosine

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CF and non-CF pig pancreas pieces cut ~4 mm3 were placed into 4% paraformaldehyde for a minimum of 2–4 days. Tissues were processed with standard IHC protocols. Testicular hyaluronidase (H3884: Sigma Aldrich, St. Louis, MO) was used for antigen retrial as previously described [54 (link)] and blocked in blocking solution (1% BSA with 1 % goat serum and 0.5% tween 20 in PBS). Slides were incubated with primary antibody, anti-3NT (06–284: Sigma Aldrich, St. Louis, MO) overnight at 4 °C. Next, goat anti-rabbit IgG (H+L) biotinylated (BA-1000: Vector Laboratories, Burlingame, CA) secondary antibody was added for 40 min followed by additional of avidin-biotin complex with a ABC-HRP kit (PK-4000: Vector Laboratories, Burlingame, CA) for 30 min. Samples were developed with a DAB kit (SK-4100: Vector Laboratories, Burlingame, CA). A common color threshold was used to extract the brown staining of the DAB kit in the images and the hue saturation over each pixel in the total tissue area was averaged.
O’Malley Y., Coleman M.C., Sun X., Lei J., Yao J., Pulliam C.F., Kluz P., McCormick M.L., Yi Y., Imai Y., Engelhardt J.F., Norris A.W., Spitz D.R, & Uc A. (2022). Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas. Advances in redox research : an official journal of the Society for Redox Biology and Medicine and the Society for Free Radical Research-Europe, 5, 100040.
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2

Immunohistochemical Analysis of 3-Nitrotyrosine

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CF and non-CF pig pancreas pieces cut ~4 mm3 were placed into 4% paraformaldehyde for a minimum of 2–4 days. Tissues were processed with standard IHC protocols. Testicular hyaluronidase (H3884: Sigma Aldrich, St. Louis, MO) was used for antigen retrial as previously described [54 (link)] and blocked in blocking solution (1% BSA with 1 % goat serum and 0.5% tween 20 in PBS). Slides were incubated with primary antibody, anti-3NT (06–284: Sigma Aldrich, St. Louis, MO) overnight at 4 °C. Next, goat anti-rabbit IgG (H+L) biotinylated (BA-1000: Vector Laboratories, Burlingame, CA) secondary antibody was added for 40 min followed by additional of avidin-biotin complex with a ABC-HRP kit (PK-4000: Vector Laboratories, Burlingame, CA) for 30 min. Samples were developed with a DAB kit (SK-4100: Vector Laboratories, Burlingame, CA). A common color threshold was used to extract the brown staining of the DAB kit in the images and the hue saturation over each pixel in the total tissue area was averaged.
O’Malley Y., Coleman M.C., Sun X., Lei J., Yao J., Pulliam C.F., Kluz P., McCormick M.L., Yi Y., Imai Y., Engelhardt J.F., Norris A.W., Spitz D.R, & Uc A. (2022). Oxidative stress and impaired insulin secretion in cystic fibrosis pig pancreas. Advances in redox research : an official journal of the Society for Redox Biology and Medicine and the Society for Free Radical Research-Europe, 5, 100040.
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3

Quantitative Analysis of 3-Nitrotyrosine

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For the analysis of total 3-NT 10 μl of sample homogenate was incubated with 10 μl of Laemmli buffer containing 0.125 M Tris base pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol. The resulting samples (250 ng per well) were loaded onto a nitrocellulose membrane with a slot-blot apparatus under vacuum pressure. The membrane was blocked for 2 h with a solution of 3% (w/v) bovine serum albumin in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 and incubated respectively with primary antibodies anti-3NT (#SAB5200009 Sigma-Aldrich, St. Louis, MO, USA) for 2h at RT. Membranes were washed and incubated with anti-rabbit or mouse IgG alkaline phosphatase secondary antibodies (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. The membrane was developed with Sigma fast tablets (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate [BCIP/NBT substrate], Sigma-Aldrich, St Louis, MO, USA). Membranes were dried and the image was acquired using ChemiDoc XP image system and analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).
Tramutola A., Abate G., Lanzillotta C., Triani F., Barone E., Iavarone F., Vincenzoni F., Castagnola M., Marziano M., Memo M., Garrafa E., Butterfield D.A., Perluigi M., Di Domenico F, & Uberti D. (2018). Protein nitration profile of CD3+ lymphocytes from Alzheimer disease patients: Novel hints on immunosenescence and biomarker detection. Free radical biology & medicine, 129.
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4

Mitochondrial Aconitase Activity Assay

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Oxidative damage to mitochondria (decreased aconitase activity) was assessed as we described [3 (link)]. Frozen-thawed mitochondria were mixed with the reaction buffer (50 mmol/L Tris-HCl, pH 7.4, 0.6mmol/L MnSO4, 5mmol/L Na citrate, 0.5 mmol/L nicotinamide adenine dinucleotide phosphate (NADP), 1 U/mL iso-citrate dehydrogenase) in a 96-well plate and the absorbance changes at 340nm were followed for 10 minutes with a plate reader (Tecan Infinite M200, San Jose, CA, USA). The aconitase activity was expressed in mU per minute per mg of mitochondrial protein. Aconitase activity was expressed in mU/min/mg of mitochondrial protein. Oxidative proteins damage in the brain was evaluated by detection of 3-nitrotyrosine (3-NT) using western blot (anti-3NT, 1:1000; Millipore).
Sosunov S.A., Ameer X., Niatsetskaya Z.V., Utkina-Sosunova I., Ratner V.I, & Ten V.S. (2015). Isoflurane Anesthesia Initiated at the Onset of Reperfusion Attenuates Oxidative and Hypoxic-Ischemic Brain Injury. PLoS ONE, 10(3), e0120456.
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5

Comprehensive Kidney Protein Analysis

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Western blot was performed using kidney cortex as described in our previous study [25 (link)]. The primary antibodies were anti-KEAP1 (Santa Cruz Biotechnology, Dallas, TX, USA; 1:1,000), anti-NRF2 (Santa Cruz Biotechnology; 1:1,000), anti-Histone H3 (Santa Cruz Biotechnology; 1:500), anti-4-HNE (Alpha Diagnostic, San Antonio, TX, USA; 1:3, 000), anti-3-NT (Millipore, Temecula, CA, USA; 1:1,000), anti-TGF-β1 (Cell Signaling, Beverly, MA, USA; 1:500), anti-COL4 (Abcam, Cambridge, MA, USA, 1:500), anti-FN (Santa Cruz Biotechnology; 1:500), anti-Smad7 (Santa Cruz Biotechnology; 1:1,000), anti-Smad3 (Santa Cruz Biotechnology; 1:1,000), anti-p-Smad3 (Cell Signaling; 1:500), anti-p-JNK (Cell Signaling; 1:500), anti-PDCD4 (Santa Cruz Biotechnology; 1:1,000), anti-t-JNK (Cell Signaling; 1:1,000), anti-Actin (Santa Cruz Biotechnology; 1:2,000) and anti-GAPDH (Santa Cruz Biotechnology; 1:3,000). These antibodies were routinely validated when they arrived from suppliers with previous positive tissues that had been defined either based on the knockout or overexpression of the target protein.
Wu H., Kong L., Tan Y., Epstein P.N., Zeng J., Gu J., Liang G., Kong M., Chen X., Miao L, & Cai L. (2016). C66 ameliorates diabetic nephropathy in mice by both upregulating NRF2 function via increase in miR-200a and inhibiting miR-21. Diabetologia, 59(7), 1558-1568.
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6

Western Blot Analysis of Protein Expression

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To analyze the protein expression, western blot analysis was performed as previously described.47 (link), 49 (link) The primary antibodies to Nrf2, HDAC1, SIRT1, SIRT6, and AMPK were purchased from Abcam (Cambridge, MA, USA). Anti-LKB1 was obtained from Sigma-Aldrich (Sigma, MO, USA), anti-HO-1, anti-NQO-1, anti-CAT, anti-IKK, anti-IκBα, anti-β-Klotho, anti-CTGF, and anti-TGF-β were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p65, anti-caspase-8, anti-caspase-3, anti-PARP, anti-Bax, anti-Bcl-2, anti-FGFR1, and anti-acetylated-lysine were purchased from Cell Signaling (Danvers, MA, USA). Anti-3-NT was from Millipore (Billerica, CA, USA), and anti-4-HNE was from Alpha Diagnostic International (San Antonio, TX, USA). After three washes with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the membrane was reacted with appropriate secondary antibodies for 1 h at room temperature. Finally, the probed proteins were stained with enhanced chemiluminescence reagent and visualized using the BIO-RAD ChemiDoc Touch Imaging System (BIO-RAD, Hercules, CA, USA).
Wang S., Wang Y., Zhang Z., Liu Q, & Gu J. (2017). Cardioprotective effects of fibroblast growth factor 21 against doxorubicin-induced toxicity via the SIRT1/LKB1/AMPK pathway. Cell Death & Disease, 8(8), e3018-.
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7

Western Blot Analysis of Oxidative Stress Markers

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For Western blots, 30 µg of proteins (CTR and DS) were separated by 12% and 7.5% SDS–PAGE using Criterion Gel TGX and blotted into a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Before blotting the gel image analyzed for total protein load was acquired to normalize blot analysis. Membranes were blocked with 3% bovine serum albumin in T-TBS and incubated for 90 min at room temperature with primary antibodies: anti-Bach1, anti-ubiquitin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-poly-ubiquitin (K63-linkage-specific), anti-HO-1, anti-HO-2 (Enzo Life Science, Farmingdale, NY, USA) anti-NQO1, anti-BVR-A (Sigma- Aldrich, St Louis, MO, USA), anti-β-actin and anti-3NT (Millipore Billerica, MA, USA). After three washes with T-TBS, the membranes were incubated for 60 min at room temperature with anti-rabbit/mouse IgG (1:5000; Sigma–Aldrich, St Louis, MO, USA) secondary antibody conjugated with horseradish peroxidase. Membranes were developed with the Super Signal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) acquired with Chemi-Doc MP (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab software (Bio-Rad, Hercules, CA, USA) that permit the normalization of a specific protein signal with the β-actin signal in the same lane or total proteins load.
Domenico F.D., Pupo G., Mancuso C., Barone E., Paolini F., Arena A., Blarzino C., Schmitt F.A., Head E., Butterfield D.A, & Perluigi M. (2015). Bach1 overexpression in Down syndrome correlates with the alteration of the HO-1/BVR-A system: insights for transition to Alzheimer Disease. Journal of Alzheimer's disease : JAD, 44(4), 1107-1120.
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8

Western Blot Analysis of Testis Oxidative Stress Markers

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Western blot was performed using testis tissue as described in our previous study [38 (link)]. The primary antibodies included anti-3-NT (Millipore, Temecula, CA, USA; 1 : 1,000), anti-4-HNE (Alpha Diagnostic, San Antonio, TX, USA; 1 : 3,000), anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 2,000), anti-ATF4 (Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti-Bax (Cell Signaling Technology, 1 : 1000), anti-Bcl-2 (Santa Cruz Biotechnology, 1 : 2,000), anti-caspase-3 (Cell Signaling Technology, 1 : 1000), anti-caspase-8 (Cell Signaling Technology, 1 : 1000), anti-caspase-12 (Cell Signaling Technology, 1 : 1000), anti-CHOP (Cell Signaling Technology, 1 : 1000), anti-Histone H3 (Santa Cruz Biotechnology; 1 : 500), anti-IL-6 (Cell Signaling Technology, 1 : 1000), anti-NRF2 (Santa Cruz Biotechnology, 1 : 1000), anti-TNF-α (Abcam, 1 : 2,000), and anti-VCAM-1 (Santa Cruz Biotechnology, 1 : 500)
Wang Y., Wu H., Xin Y., Bai Y., Kong L., Tan Y., Liu F, & Cai L. (2017). Sulforaphane Prevents Angiotensin II-Induced Testicular Cell Death via Activation of NRF2. Oxidative Medicine and Cellular Longevity, 2017, 5374897.
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