Background Proteins S-acylation (also known as palmitoylation) is the reversible post-translational addition of acyl lipids to cysteine residues in proteins through a thioester relationship. account for the observed membrane association of flower microtubules. As alpha-tubulins are ubiquitously indicated they can potentially be used like a positive control for the S-acylation assay regardless of the cell type under study. Conclusion We provide a powerful biotin switch protocol that allows the quick assay of protein S-acylation state in vegetation using standard laboratory techniques and without the need for expensive or SU 11654 specialised products. We propose alpha-tubulin as a useful positive control for the protocol. Background S-acylation is definitely a reversible post-translational changes of cysteine residues in proteins. It is also known as protein palmitoylation but this term is definitely less accurate because a variety of acyl lipid organizations (e.g. palmitoyl SU 11654 stearoyl) can be added to cysteine residues. S-acylation is sometimes puzzled with acetylation which is the addition of acetyl organizations to amines but the two are fundamentally different processes. S-acyl organizations associate with membranes and are frequently found on proteins associated with membrane microdomains [1 2 There have been three positive identifications of S-acylated flower proteins. OsCPK2 a protein kinase from rice was labelled by H3-palmitic acid in vivo [3]. ROP10 a type II small GTPase was revised by H3-palmitoyl-CoA in vitro [4] and ROP6 a type I small GTPase was SU 11654 shown to be primarily revised by stearic acid by gas chromatography [5]. Additional proteins such as RIN4 [6] AGG2 [7 8 and various calcium-dependent [9] and calcium-sensing kinases [10] have been predicted to be S-acylated based on mutational studies (replacing relevant cysteine residues) checks for membrane association and the use of S-acylation inhibitors but no direct evidence exists for his or her S-acylation. Computational methods for predicting S-acylation sites have been developed such as Terminator2 [11 12 which efforts to identify S-acylation sites within the 1st 20 amino acids of a protein and CSS-Palm [13] and NBA-Palm [14] which aim to determine S-acylation sites throughout a protein. As yet no biochemical validation of previously unfamiliar S-acylated proteins becoming recognized using these programs has been reported in the literature. Once potential S-acylation sites have been recognized either through predictive or experimental means a method for verification of the site is required. A novel method for identifying S-acylation sites in mammals was developed [15] termed the biotin switch protocol which allowed accurate and specific assaying of S-acylation sites but did not work reliably in all situations. Adaptations and revisions of this method possess since been developed and applied to budding candida [16 17 and vegetation (this study) and are reliable and work well with all proteins without further optimisation. The method described here is equally relevant for the detection of S-acylation of native or transgenic proteins from flower tissue. This is an advance on previous methods which either require large amounts of starting material expensive chromatographic and mass spectroscopy equipment [5] or incubation of cell cultures or heterologously expressed proteins in the presence of H3-palmitic acid followed by immunoprecipitation and autoradiography [18]. This latter method is expensive hazardous and extremely time consuming typically requiring upward of 6 weeks for exposure of the autoradiograph. Here we present modifications and refinements of the biotin switch method [15] and a detailed protocol to allow rapid and affordable identification of S-acylation sites in plant proteins. This protocol c-COT represents an improvement over the standard assays for S-acylation as it does not involve the use of radioactive chemicals addition of exogenous chemicals for labelling or pharmacological treatments commonly used to aid in label incorporation. We have used the biotin switch assay to show that the α-subunits of Arabidopsis tubulin are S-acylated. As α-tubulin is ubiquitously expressed and anti-α-tubulin antibodies are readily available it can be used as an internal control during biotin switch acylation assays SU 11654 without any extra processing of protein samples. We have also assayed the S-acylation state of the heterotrimeric G-protein γ 2 subunit AGG2. It has been suggested that AGG2 might be S-acylated because AGG2 mutants lacking a cysteine residue in the C terminus are incorrectly localised in vegetable cells [7 8 Outcomes The biotin change technique summarised in.