By Ganesh K. Agrawal, Jay J. Thelen (auth.), Marjo de Graauw PhD (eds.)
Protein phosphorylation controls many uncomplicated mobile tactics, resembling telephone development, differentiation, migration, metabolism, and telephone demise, and its learn gives you key insights into the sign transduction pathways which are activated in cells according to varied stimuli, comparable to development issue stimulation or publicity to toxicants. In Phospho-Proteomics: tools and Protocols, professional researchers give a contribution either well-established protocols and a few of the latest options for the id and review of protein phosphorylation on Tyr, Ser, and Thr residues, together with issues equivalent to 2-dimensional gel electrophoresis and protein phosphorylation, enrichment of phospho-proteins and peptides, quantitative research of phosphorylation through labeling and MS research, and antibody and kinase arrays. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters contain short introductions to their respective topics, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and notes on troubleshooting and keeping off recognized pitfalls.
Comprehensive and state of the art, Phospho-Proteomics: equipment and Protocols is a perfect reference for either new and skilled scientists who desire to achieve perception into the present advancements within the box and to discover idea to pursue its future.
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Additional info for Phospho-Proteomics: Methods and Protocols
11. Briefly spin the samples, collect the supernatant and transfer to new siliconised tubes. Add sufficient 50% AcN/5% TFA to cover the gel pieces and shake for 20–30 min to aid peptide extraction. Remove the supernatant and pool together with the first. Repeat this step twice. 12. Speed vac to dryness. Samples can be stored at −20°C or be directly analysed by MS. 13. 1% FA by gently shaking. 2. Protein Identification by LC-MS/MS 1. Chromatographic separation is accomplished by injecting 5 μL of digested peptide samples onto a reversed phase capillary column (PepMap, 75 μm × 150 mm) using a nanoflow HPLC system (Dionex, Ultimate) connected on-line to an ESI Q-TOF mass spectrometer (Waters).
Wash the IMAC column using 40 μL of IMAC loading buffer. Enrichment and Characterization of Phosphopeptides by Immobilized Metal Affinity 51 Fig. 1. The principle of making IMAC micro-columns for a complex sample. The constricted end of a 200-μL GELoader tip is squeezed to prevent the IMAC beads from leaking. This is done by pressing the tip of a pen onto the end of the GELoader tip (a). Cut the top of the GELoader tip to make a plastic syringe fit into the opening (b). Load the IMAC beads onto the inside of the GELoader tip (c).
See Fig. 1). Fig. 1. Equalising intensities of labelling media. B, decreasing amounts but equal volumes of each radioisotope (starting intensity, ∼5 nCi; bottom line) were spotted onto Whatman poly-ethyleneimine-impregnated p81 paper. After drying, the paper was imaged for 24 h without (left) or with (right) two acetate sheets placed between the paper and the screen; the difference in intensities at this time was about 1:3 (32P:33P). Hence three times more 33P isotope compared with 32P isotope needs to be used for in vivo labeling.