In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique
通过生物素转换技术对重组蛋白中的S酰化作用进行体外检测
发布: 2014年11月20日第4卷第22期 DOI: 10.21769/BioProtoc.1296 浏览次数: 9588
评审: Zhaohui LiuAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2014
Abstract
Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described an easy protocol for quick in vitro detection of protein S-acylation using the Arabidopsis protein kinase, PBS1, as an example. To determine whether PBS1 is modified through thioester linkage to acyl groups, we employed a “biotin switch” assay (Hemsley et al., 2008). This work was first published in Qi et al. (2014), but we expand on the method here. PBS1 functions within the basal immune system of plants, and is a target of the bacterial cysteine protease, AvrPphB (Shao et al., 2002; Zhang et al., 2010). It contains a predicted N-terminal S-acylation motif (MGCFSCFDS), with both Cys-3 and Cys-6 residues predicted to be palmitoylated by CSS-Palm 3.0 (http://csspalm.biocuckoo.org/; Ren et al., 2008). Our method utilizes hydroxylamine-induced cleavage of thioester bonds, which results in free sulfhydryl groups that can then be conjugated to a biotin derivative, 1-biotinamido-4-[4′-(maleimidomethyl) cyclohexanecarboxamido]-butane (Biotin-BMCC). The conjugates are detectable by Western blot with streptavidin-horseradish peroxidase. The whole process of in vitro labelling and detection took less than 3 days, allowing the fast detection of protein modifications via thioester bonds such as palmitoylation.
Materials and Reagents
- Nicotiana benthamiana (N. benthamiana) plants
- Agrobacterium tumefaciens strain GV3101 (pMP90)
- Bacto yeast extract (BD Biosciences, catalog number: 288620 )
- Bacto tryptone (BD Biosciences, catalog number: 211699 )
- Magnesium chloride hexahydrate (EMD Millipore, catalog number: 442611 )
- Acetosyringone (Sigma-Aldrich, catalog number: D134406-5G )
- Dexamethasone (Sigma-Aldrich, D4902-1G )
- Trizma Tris base (Sigma-Aldrich, catalog number: 93362 )
- Sodium chloride (EMD Millipore, catalog number: 567442 )
- Nonidet P-40(Sigma-Aldrich, catalog number: 21-3277 )
- Plant proteinase inhibitor cocktail (Sigma-Aldrich, catalog number: P9599-5M )
- SDS (Sigma-Aldrich, catalog number: L3771-100G )
- Glycerol (EMD Millipore, catalog number: 356350 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148-25ML )
- EDTA (Sigma-Aldrich, catalog number: E6758-100G )
- BSA (Sigma-Aldrich, catalog number: A4503-100G )
- Bromphenol blue (EMD Millipore, catalog number: BX1410 )
- Anti-HA monoclonal antibody matrix (Roche Diagnostics, catalog number: 11867423001 )
- Tris-HEPES-SDS polyacrylamide gels (Thermo Fisher Scientific, catalog number: 25204 )
- Anti-HA peroxidase (Sigma-Aldrich, catalog number: H6533-1VL )
- High Sensitivity Streptavidin-HRP Conjugate (Thermo Fisher Scientific, catalog number: 21130 )
- ImmunoStar HRP Substrate Kit (Bio-Rad Laboratories, catalog number: 170-5070 )
- N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876-5G )
- Hydroxylamine (Sigma-Aldrich, catalog number: 467804-10ML )
- 1-biotinamido-4-[4′(maleimidomethyl)-cyclohexane-carboxamido]-butane (EZ-link BMCC-Biotin) (Thermo Fisher Scientific, catalog number: 21900 )
- Nitrocellulose Membrane (Thermo Fisher Scientific, catalog number: WP4HY00010 )
- Metro-Mix 360 (Sun Gro Horticulture Canada)
- LB liquid medium (see Recipes)
- Lysis buffer (see Recipes)
- 4x SDS loading buffer (see Recipes)
- 5x nonreducing protein sample buffer (see Recipes)
Equipment
- Confocal microscope system (Leica Microsystem, model: TCS SP5 )
- Mini-Protean Electrophoresis and Blotting system (Bio-Rad Laboratories)
- Benchtop Centrifuge 5424 (Eppendorf)
- Lab tube rotator (Thermo Fisher Scientific)
- Gene Pulser (Bio-Rad Laboratories, catalog number: 1652076 )
- 1 ml syringe (BD Biosciences, catalog number: 305945 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Qi, D. and Innes, R. W. (2014). In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique. Bio-protocol 4(22): e1296. DOI: 10.21769/BioProtoc.1296.
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Qi, D., Dubiella, U., Kim, S. H., Sloss, D. I., Dowen, R. H., Dixon, J. E. and Innes, R. W. (2014). Recognition of the protein kinase AVRPPHB SUSCEPTIBLE1 by the disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 is dependent on S-acylation and an exposed loop in AVRPPHB SUSCEPTIBLE1. Plant Physiol 164(1): 340-351.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 修饰
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