Tissues and cells were lysed in RIPA buffer (cat#V900854, Sigma, MO, USA) and protein concentration was measured with the BCA method (cat#P0012, Beyotime, Shanghai, China). Proteins were isolated by SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, USA), incubated with primary and secondary antibodies and imaged with the ECL detection reagent (cat#12630, CST, California, USA) using a ChemiDoc™ XRS+ System. The primary antibodies included anti-Mus81 (1:1,000; cat#ab14387, Abcam, Cambridge, UK), anti-BRD4 (1:1,000; cat#ab128874, Abcam), anti-ZEB1 (1:1,000; cat#ab180905, Abcam), anti-E-cadherin (1:1,000; cat#3195, CST, California, USA), anti-N-cadherin (1:1,000; cat#13116, CST), anti-Snail1 (1:1,000; cat#9782, CST), anti-ZEB2 (1:1,000; cat#14026-1-AP, Proteintech, Wuhan, China), anti-Sirt5 (1:500; cat#15122-1-AP, Proteintech), anti-GAPDH (1:5,000; cat#G9545, Sigma). The secondary antibodies included HRP conjugated goat anti-Rabbit (1:3,000; SA00001-15, Proteintech) and anti-Mouse (1:3000; cat# SA00001-1, Proteintech).
Anti sirt5
Manufactured by Proteintech
Sourced in United States
Anti-SIRT5 is a specific antibody that recognizes the SIRT5 protein. SIRT5 is a member of the sirtuin family of enzymes, which play a role in cellular processes such as metabolism and stress response. The anti-SIRT5 antibody can be used to detect and study the SIRT5 protein in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti brd4, by Abcam (1 mentions)
Anti zeb1, by Abcam (1 mentions)
Anti mus81, by Abcam (1 mentions)
Anti zeb2, by Proteintech (1 mentions)
Sa00001 15, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Bca method, by Beyotime (1 mentions)
Anti β actin primary antibody, by Cell Signaling Technology (1 mentions)
Anti sirt3, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
U rflt50 power supply unit, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Bullet blender homogenizer, by Next Advance (1 mentions)
5 protocols using anti sirt5
1
Protein Expression Analysis by Western Blotting
2
Western Blot Analysis of Sirtuins
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Cells and mice tissue were lysed in RIPA buffer with PMSF (Beyotime, Shanghai, China), and then protein was adjusted to the same level, added loading buffer and boiled at 100 °C for 5 min. A 50µg mass of protein lysates were loaded on 10% SDS-PAGE gels and transferred using PVDF membranes. After block, the membranes were incubated overnight with anti-SIRT3 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT4 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT5 (1:1000; Proteintech, Wuhan, Hubei, China) or anti-β-actin primary antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA), followed by incubation with appropriate secondary antibodies (1:10,000; Cell Signaling Technology, MA, USA) at 37 °C for 1h. The signal was visualized and captured with a ChemiDoc XRS+system (Bio-Rad, Hercules, CA, USA).
3
Immunohistochemical Analysis of SIRT5 Expression
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Mice were anesthetized with an overdose of choral hydrate and with the cervical dislocation execution. The right ankle joint of mice was taken and samples collected were decalcified in 13% EDTA (pH 7.3), then placed in 30% sucrose overnight and embedded in OCT at −20 °C the next day. Frozen sections were cut (30 μm) and placed on glass micro slides coated with APS. Sections were fixed in 4% paraformaldehyde and incubated in a blocking solution containing 3% BSA, 0.02% Na N30.1%, and Triton X-100 in PBS for 2h at room temperature. After blocking, sections were incubated with the appropriate primary antibodies in a blocking solution at 4 °C overnight. The primary antibodies used were: anti-SIRT5 (1:400) from ProteinTech. The secondary antibody was a goat anti-rabbit (1:500) antibody (ProteinTech Group, Chicago, IL, USA). Slides were mounted with coverslips and visualized using a fluorescence microscope (CKX41 with an Olympus U-RFLT50 Power Supply Unit; Olympus, Tokyo, Japan). Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was applied to analyze the integrated optic density (IOD) and pixel area (AREA), and calculated the average optical (AO), AO = IOD/AREA.
4
Protein Extraction and Western Blot Analysis
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The tissues were immediately excised to extract proteins. Sampled proteins were prepared by adding a lysis buffer containing 250 mM NaCl, 50 mM Tris–HCl pH 7.4, 50 mM sodium F, 1%NP-40, 0.02%Na N3, 5 mM EDTA, 1 mM Na3VO4, and 1 × protease inhibitor cocktail (AMRESCO, Solon, OH, USA). After homogenization using a Bullet Blender homogenizer (Next Advance, NY, USA), the extracted proteins (30 μg per sample assessed by BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-T buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), incubated with the primary antibody in TBS-T with 1% bovine serum albumin for 1 h at room temperature. After peroxidase-conjugated secondary antibody (1:5000) was used, the image intensities of specific bands were quantified using ImageJ software (Bethesda, MD, USA). Antibodies used in the experiments were: anti-β-ACTIN (ProteinTech Group, Chicago, IL, USA), and anti-SIRT5 (ProteinTech Group, Chicago, IL, USA).
5
Co-IP Validation of Protein Interactions
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Co-IP experiments were performed to verify the binding between proteins of interest in NP cells. The co-IP complexes were purified by a MilliporeSigmaTM PureProteomeTM Protein A/G Mix Magnetic Bead System (#LSKAGAG10, Fisher Scientific, USA). The dosage of the IP antibody was 5 µg of anti-AIFM1 (Santa Cruz Biotechnology, sc-13116, USA) and 4 µg of anti-SIRT5 (Proteintech, 15122-1-AP, China). The proteins were harvested and analyzed by Western blot analysis.
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