Sufficient amounts of anti-SOD2 or anti-Sirt3 antibody (Santa Cruz) were added to the fresh protein supernatant and incubated at 4°C overnight. Next, 25 µl of protein A+G agarose beads (Beyotime, Haimen, China) was added and incubated for 3 h at 4°C. The mixture was then centrifuged at 2500× g for 5 min at 4°C°C, and the supernatant was discarded. The precipitate was washed five times with ice-cold PBS. After washing, the immunocomplex was boiled in 2x SDS buffer for 5 min. The supernatant was then collected by centrifugation and subjected to western blotting analyses with anti-acetyl-lysine antibody (1∶1000, Cell Signaling Technology), anti-SOD1(1∶500, Santa Cruz), anti-SOD2 (1∶500, Santa Cruz), anti-SOD3 (1∶500, Santa Cruz), anti-Sirt3 (1∶500, Santa Cruz), anti-Sirt4 (1∶500, Santa Cruz), and anti-Sirt5 antibodies (1∶500, Santa Cruz). The control for these IP experiments was normalized against rabbit IgG.
Anti sirt3 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-Sirt3 antibody is a research tool used to detect the presence and expression levels of the Sirt3 protein in biological samples. Sirt3 is a member of the sirtuin family of proteins and plays a role in mitochondrial function and metabolism. The antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to analyze the Sirt3 protein in different cell and tissue types.
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Lab products found in correlation
Anti sod2, by Santa Cruz Biotechnology (1 mentions)
Anti sirt3, by Santa Cruz Biotechnology (1 mentions)
Anti sod1, by Santa Cruz Biotechnology (1 mentions)
Anti acetyl lysine antibody, by Cell Signaling Technology (1 mentions)
Protein a g agarose bead, by Beyotime (1 mentions)
Anti sod3, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti ampk, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Anti rabbit pgc 1α, by Cell Signaling Technology (1 mentions)
Anti rabbit p ampk thr 172, by Cell Signaling Technology (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Goat anti guinea pig igg, by Thermo Fisher Scientific (1 mentions)
Gfap antibody, by Merck Group (1 mentions)
Neun antibody, by Merck Group (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Optical microscope, by Olympus (1 mentions)
5 protocols using anti sirt3 antibody
1
Immunoprecipitation and Western Blot Analysis
2
Immunoblotting Analysis of Mitochondrial Regulators
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Primary antibodies including anti-rabbit PGC-1α, anti-rabbit p-AMPK (thr 172); anti-rabbit AMPK (Cell Signaling Technology, Inc., Danvers, MA, USA); Anti-SIRT3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-rabbit TFAM, anti-rabbit NRF-1 and anti-rabbit GAPDH were obtained from Abcam Biotechnology (Cambridge, MA, USA). Horse-radish peroxidase-conjugated anti-rabbit IgG (Cell Signaling) and enhanced chemiluminescence reagents (ECL and ECL plus) were obtained from Amersham Biosciences (Buckinghamshire, UK). All other reagents were obtained from Sigma (St. Louis, MO).
3
Sirt3 Immunolocalization in Rat Auditory Cortex
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Using an immunofluorescence method and a laser-scanning confocal microscope (Nikon, Japan), we detected the distribution of Sirt3 in the auditory cortex. Thirty-six rats (n = 6 per subgroup) were transcardially perfused with saline followed by 4% paraformaldehyde solution. The brains were removed and postfixed in 4% paraformaldehyde overnight at 4°C. A 5-µm section was deparaffinized in xylene and rehydrated through graded concentrations of ethanol. Following antigen retrieval, nonspecific binding was blocked with donkey serum albumin for 1 h at room temperature. Next, the anti-Sirt3 antibody (diluted 1∶50, Santa Cruz) was added and incubated overnight at 4°C. After three washes with PBS, the sections were incubated for 1 h with fluorescently tagged secondary antibody. Control staining was performed in the absence of primary antibody.
4
Immunofluorescence Protocol for Evaluating SIRT3 Expression in Neurons and Astrocytes
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Immunofluorescence was carried out based on standard protocol. Tissue blocks containing the temporal cortex were ordinarily fixed, dehydrated, and cut into 6 μm thick sections. The sections were first cocultured with 5% normal fetal bovine serum in PBS including 0.1% Triton X-100 for 2 h and then with anti-neuron-specific nuclear protein (NeuN) antibody (1 : 200, Millipore, USA) and anti-SIRT3 antibody (1 : 100) or anti-glial fibrillary acidic protein (GFAP) antibody (1 : 200, Millipore, USA) and anti-SIRT3 antibody (1 : 100, Santa Cruz Biotechnology Inc., USA) overnight at 4°C. Secondary antibodies included goat anti-rabbit IgG (1 : 200) and goat anti-guinea pig IgG (1 : 200, Invitrogen Life Technologies, USA). The sections were incubated with secondary antibodies and counterstained with DAPI counterstain. The fluorescence images were acquired using Nikon TE200 (Nikon Corporation Company, Tokyo, Japan) with a Spot RT digital camera.
5
Immunohistochemical Analysis of Sirt3 in ICH
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Immunohistochemistry was performed 24 h following ICH using a universal streptavidin-perosidase (SP) kit (Zhongshanjinqiao Biotechnology, Beijing, China) according to the manufacturer's instructions. Briefly, after deparaffinization and rehydration, sections were incubated overnight at 4°C with anti-Sirt3 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Samples without primary antibodies were used as a negative control. Subsequently, the sections were incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h. Finally, the sections were stained with diaminobenzidine and counterstained with hematoxylin. Three microscope fields for each section were randomly selected for photographing under an optical microscope (Olympus). All images were examined in a blinded manner.
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