棕榈酸治疗的人肾小管上皮细胞中乙酰赖氨酸的免疫沉淀和长链酰基辅酶A脱氢酶和β-羟酰基辅酶A脱氢酶的蛋白质印迹

Tingting  Lv; Suwei  Zhu; Yuan  Ma; Hong  Feng; Qiang Wan

doi:10.21769/BioProtoc.3765

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells

棕榈酸治疗的人肾小管上皮细胞中乙酰赖氨酸的免疫沉淀和长链酰基辅酶A脱氢酶和β-羟酰基辅酶A脱氢酶的蛋白质印迹

QW Qiang Wan email

发布: 2020年09月20日第10卷第18期 DOI: 10.21769/BioProtoc.3765 浏览次数: 4113

评审: Xiaoyi ZhengYing ShiJake J. Wen

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Physiology and Biochemistry, featuring study using the protocol.

Nov 2019

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

Boyden小室法侵袭实验的吸光度检测实验方案

Kathleen C. Brown [...] Piyali Dasgupta

2024年08月05日 1001 阅读

哺乳动物线粒体和胞质顺乌头酸酶的凝胶内活性测定——分区特异性氧化应激与铁状态的替代标志物

Wing-Hang Tong and Tracey A. Rouault

2024年12月05日 714 阅读

氧肟印迹法实现羰基化蛋白的可靠灵敏检测

Filip Luka Mikulić [...] Mladen Merćep

2025年08月05日 411 阅读

Abstract

As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial cells). The scheme provides the readers with clear steps so that this method could be applied to detect the acetylation level of various proteins.

Keywords: Immunoprecipitation (免疫沉淀反应)

Acetylation (乙酰化作用)

LCAD (长链酰基辅酶A脱氢酶)

Beta-HAD (β-羟酰基辅酶A脱氢酶)

Palmitic acid (PA) (棕榈酸)

HK-2 cells (HK-2 细胞)

Background

Post-translational modifications (PTMs) enable the cell with a highly dynamic mechanism for regulation of cellular pathways (Zhao et al., 2010). Acetylation has emerged as one of the major post-translational protein modifications. Accumulating evidences indicate that acetylation rivals phosphorylation as a regulatory modification in mitochondria (Henriksen et al., 2012). Over 60% of mitochondrial proteins are acetylated, being of notice, which were involved in energy metabolism such as tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), fatty acid oxidation and amino acid metabolism (Hirschey et al., 2010; Hebert et al., 2013). Acetylation is also considered to be one of the main adaptive mechanisms for mitochondria in sensing nutritional challenges. Under the condition of excess energy supplement, mitochondrial proteins are hyperacetylated. Insufficient energy availability leads to a decreased acetylation level of mitochondrial proteins (Schwer et al., 2009). A large number of mitochondrial metabolic enzymes are acetylated in diabetes, including the FAO enzymes LCAD and beta-HAD (Kosanam, 2014). Dharendra et al. indicated that the upregulation of acetylation status of mitochondrial fatty acid oxidation enzymes promoted fatty acid oxidation in the heart of high fat diet mice (Thapa et al., 2018). Our previous study also found that the acetylation levels of FAO enzymes LCAD and β-HAD were enhanced in HK-2 cells treated with palmitic acid (PA) (Lv et al., 2019). Protein lysine acetylation refers to covalent addition of acetyl group to lysine residue (N-lysine acetylation). At present, since there are no acetylated antibodies of LCAD and HAD, the detection of LCAD and HAD acetylation levels mainly depends on immunoprecipitation analysis which mainly concentrates the acetylated proteins by using acetyl-lysine antibody and then detects the content of acetylated LCAD and acetylated HAD by using LCAD antibody and HAD antibody respectively (Figure 1).

Figure 1. Flowchart of IP

Materials and Reagents

100 mm x 20 mm cell culture dishes (Coring, catalog number: 430167 )
1.5 ml microcentrifuge tube
Disposable syringe filters (Millipore, catalog number: SLGP033RB )
Polyvinylidene fluoride (PVDF) Transfer Membrane (Thermo Scientific, catalog number: 88518 )
HK-2 cells (ATCC, catalog number: CRL-2190 )
Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific, catalog number: 26147 )
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number: 11885084 )
Fetal bovine serum (FBS) (Gibco, catalog number: 10099141C )
Palmitic acid (PA) (Sigma, catalog number: P5585 )
Albumin Bovine (BSA) (Solarbio, catalog number: A8850 )
Halt Protease and Phosphatase inhibitor cocktail (Thermo Scientific, catalog number: 78442 )
Phosphate buffered saline (PBS) (Solarbio, catalog number: P1020 )
Mouse-Acetyl-lysine antibody (Abcam, catalog number: ab22550 )
Rabbit-LCAD antibody (Proteintech, catalog number: 17526-1-AP )
Rabbit-beta-HAD antibody (Sigma, catalog number: HPA037539 )
Rabbit-COXIV antibody (Proteintech, catalog number: 11242-1-AP )
Secondary anti-rabbit IgG HRP-conjugated antibody
Glycine (Solarbio, catalog number: G8200 )
Sodium dodecyl sulfate (SDS) (Solarbio, catalog number: S8010 )
Ammonium persulfate (APS) (Solarbio, catalog number: A8090 )
N,N,N',N'-Tetramethylethylenediamine (TEMED) (Invitrogen, catalog number: 15524010 )
Tween-20 (Solarbio, catalog number: T8220 )
Tris (Solarbio, catalog number: T8060 )
1.5 M Tris(hydroxymethyl)aminomethane (Tris-HCl) (pH 8.8) (Solarbio, catalog number: T1010 )
1.0 M Tris(hydroxymethyl)aminomethane (Tris-HCl ) (pH 6.8) (Solarbio, catalog number: T1020 )
30% Acrylamide (Solarbio, catalog number: A1010 )
Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, catalog number: P0012S )
Immobilon Western Chemiluminescent HRP Substrate (Millipore, catalog number: WBKLS0050 )
Methanol (Tianjin Kaitong, China)
Sodium chloride (Tianjin Kaitong, China)
12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separating gels (10 ml) (see Recipes)
SDS-PAGE stacking gels (4 ml) (see Recipes)
1x TBST (Tris-HCl, NaCl, Tween 20) (see Recipes)
2.5% BSA (see Recipes)
10 mM PA (PA stock solution) (see Recipes)
200 μM PA (see Recipes)

Equipment

Centrifuge (Eppendorf, catalog number: 5810R )
P1000 pipette (Eppendorf, catalog number: 3111000858 )
P200 pipette (Eppendorf, catalog number: 3111000840 )
P20 pipette (Eppendorf, catalog number: 3111000823 )
P10 pipette (Eppendorf, catalog number: 3111000815 )
Rotator
Benchtop rocker (HEIDOLPH, catalog number: Titramax 101 )
Spectrophotometer
Blot and gel imager (General Electric Company, catalog number: Amersham Imager 680 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Lv, T., Zhu, S., Ma, Y., Feng, H. and Wan, Q. (2020). Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells. Bio-protocol 10(18): e3765. DOI: 10.21769/BioProtoc.3765.