Islets or INS-1 cells were collected in lysis buffer (Cell Signaling Technology). Protein lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (BioRad). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies. Anti-ECHA and anti-SIRT4 was from Abcam. Anti-SIRT3, anti-SIRT5, and anti-rabbit IgG conjugated with HRP were from Cell Signaling Technology. Anti-α-tubulin was from Proteintech. Anti-Flag was from Sigma-Aldrich. Anti-acetyllysine was from PTM Biolab. Immunoprecipitation was performed by incubating protein lysates with FLAG M2 Affinity Gel (Sigma) or ECHA antibody for 2 h and then with Protein A/G PLUS-Agarose (Santa-Cruz) overnight at 4 °C. The binding complexes were washed and then eluted with loading buffer. Standard western blotting was followed using anti-acetyllysine antibody for acetylation analysis.
Anti sirt4
Manufactured by Abcam
Sourced in United States
Anti-SIRT4 is a protein that functions as an enzyme involved in the regulation of cellular processes. It plays a role in energy metabolism and mitochondrial function. This product is a research-use only tool for studying the biological functions of Anti-SIRT4.
Automatically generated - may contain errors
Lab products found in correlation
Anti sirt5, by Cell Signaling Technology (2 mentions)
Anti sirt3, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (1 mentions)
Flag m2 affinity gel, by Merck Group (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Goat anti rabbit igg h l, by Abcam (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Goat anti mouse igg h l, by Abcam (1 mentions)
Anti α tubulin, by Beyotime (1 mentions)
Pan acetyllysine, by Cell Signaling Technology (1 mentions)
Ab104836, by Abcam (1 mentions)
Ab168379, by Abcam (1 mentions)
3 protocols using anti sirt4
1
Western Blot Analysis of Acetylation
2
Western Blot Analysis of SIRT4 Protein
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Total protein was extracted from cells and tissues using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime Institute of Biotechnology, Shanghai, China). Protein determination was performed using the BCA Protein assay kit (Takara Bio, Inc., Otsu, Japan) and proteins (25 µg) were separated by SDS-PAGE (10% gels) and then transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) and blocked in 5% bovine serum albumin (Beyotime Institute of Biotechnology) was diluted in PBS for 2 h at room temperature. Membranes were incubated with anti-SIRT4 (catalog no. ab10140; 1:300, Abcam) or mouse anti-β-actin (catalog no. sc-58673, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies at 4°C overnight, followed by incubation with Rabbit Anti-goat IgG H&L (catalog no. ab6697; 1:5,000; Abcam) or goat anti-mouse IgG H&L (catalog no. ab6708; 1:5,000; Abcam) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) via FluorChem M imaging system (FCM, FM0422; ProteinSimple, San Jose, CA, USA) and analyzed using the Image J software (https://imagej.nih.gov/ij/ ).
3
Immunoblotting of Sirtuin Proteins
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Western blotting was conducted as previously described [45 , 46 ]. The primary antibodies used are as follows: anti‐Sirt3 (#5490; Cell Signaling Technology, Boston, MA, USA), anti‐Sirt4 (ab124521; Abcam, Cambridge, MA), anti‐Sirt5 (#8782; Cell Signaling Technology), anti‐α‐tubulin (Beyotime, Shanghai, China), anti‐hexokinase II (ab104836; Abcam), anti‐PDHE1α (ab168379; Abcam), anti‐SDH (ab14714; Abcam), anti‐COX‐IV (ab33985; Abcam), pan‐succinyl‐lysine (PTM‐401), pan‐malonyl‐lysine (PTM‐901), pan‐glutaryl‐lysine (PTM‐1151), pan‐acetyl‐lysine (#9441; Cell Signaling Technology) and Flag (4110‐20; Shanghai Genomics, Shanghai, China). The antibodies were purchased commercially.
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