Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining

Meisrimler, Claudia-Nicole; Schwendke, A.; Lüthje, Sabine

doi:10.3389/fpls.2015.00230

Cited by 3 publications

(3 citation statements)

References 49 publications

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“…A detailed step-by-step protocol for Phos-Tag staining is outlined elsewhere [42]. Fluorescent stains such as ProQ Diamond (Molecular Probes) or quercetin-aluminum (III)-appended complex can be used to selectively visualize phosphorylated proteins (total or following IP) following resolution on polyacrylamide gel [49, 50]. These staining methods eliminate the need for immunoblotting with phospho-specific antibodies, but are rarely used due to lack of specificity.…”

Section: Detection Of Phosphorylationmentioning

confidence: 99%

Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation

Arif

Jia

Halawani

et al. 2017

Methods

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Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of translation (GAIT) complex that directs transcript-selective translational control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is essential for its release from the parental multi-aminoacyl tRNA synthetase complex (MSC), for binding to other GAIT complex proteins, and for regulating the binding to target mRNAs. Importantly, phosphorylation is the common driving force for the context- and stimulus-dependent release, and non-canonical activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase (KARS). Here, we describe the concepts and experimental methodologies we have used to investigate the influence of phosphorylation on the structure and function of EPRS. We suggest that application of these approaches will help to identify new functional phosphorylation event(s) in other AARSs and elucidate their possible roles in noncanonical activities.

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Section: Detection Of Phosphorylationmentioning

confidence: 99%

Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation

Arif

Jia

Halawani

et al. 2017

Methods

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“…Chromatography separation can be combined with activity profiling, Western blot and MS. Recently, a protocol for phos-tag PAGE (Kinoshita and Kinoshita-Kikuta, 2011 ), which is an affinity PAGE method for phosphorylated proteins, was demonstrated for the application in targeted pea proteomics (Meisrimler et al, 2015 ).…”

Section: Isoenzyme Analysis—standard Techniques or Native Approachesmentioning

confidence: 99%

“…Not all staining methods are compatible with modified SDS-PAGE. Some proteins have a higher sensitivity for SDS and comparable detergents (Meisrimler et al, 2015 ). In these cases, native PAGE replaces the modified SDS-PAGE.…”

Section: Isoenzyme Analysis—standard Techniques or Native Approachesmentioning

confidence: 99%

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

et al. 2015

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Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

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Recombinant protein expression: Challenges in production and folding related matters

Beygmoradi

Homaei

Hemmati

et al. 2023

International Journal of Biological Macromolecules

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Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining

Cited by 3 publications

References 49 publications

Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation

Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

Recombinant protein expression: Challenges in production and folding related matters

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