By Luca Goitre, Eliana Trapani, Lorenza Trabalzini, Saverio Francesco Retta (auth.), Lorenza Trabalzini, Saverio Francesco Retta (eds.)

Featuring experimental ways that make clear the complexity of Ras GTPase organic capabilities, Ras Signaling: tools and Protocols comprises common overviews and particular purposes of either well-established and lately built examine ideas, together with biochemical, biophysical, molecular biology, genetic and behavioral techniques, complex excessive answer fluorescence and electron microscopy imaging and “omics” applied sciences. via this, the distinctive quantity presents details on expression, post-translational transformations, subcellular localization and dynamics, regulatory mechanisms of upstream and downstream signaling pathways and finally, organic actions and features of Ras GTPases in numerous version structures, together with low and high eukaryotic organisms. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters comprise short introductions, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols and pointers on troubleshooting and fending off identified pitfalls.

Wide-ranging and authoritative, Ras Signaling: tools and Protocols serves as an relief for investigators of other backgrounds and pursuits on the topic of the a number of physiological and pathological capabilities of the massive superfamily of Ras GTPases.

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This reaction mixture contains modified product and sortase as well as unreacted HVR and catalytic domain. Lane 2 shows sortase and K-Ras 4B catalytic domain SDS PAGE gel of purified fully modified K-Ras4B Lane 1 Lane 2 Fully Modified K-Ras 4B Lane 3 Sortase A K-Ras 4B catalytic domain 25 kD 15 kD Fig. 4 SDS-PAGE gel of purified fully modified K-Ras4B. The figure shows the fully modified product after components of the sortase reaction were removed using nickel affinity purification. Lane 1 contains fully modified K-Ras 4B.

3. 2 mCi/mL). Allow ethanol solvent to evaporate completely (see Note 2). 4. Add 6 mL of labeling medium on top of the 10 cm plate containing the [3H]palmitate and incubate at 37 °C for 5 min. 5. Change medium for the transfected cells using the prepared labeling medium containing [3H]palmitate. , 1 μM FK506, 50 ng/mL cycloheximide, 500 nM rapamycin, or 25 μM 2-BP. Incubate at 37 °C overnight (see Note 3). 6. The next day, place cells on ice. Manually remove medium from cells and wash once with ice-cold PBS.

The CAAX sequence of nascent Ras proteins is modified in three steps. First, a farnesyl lipid is added to the CAAX cysteine by farnesyltransferase. Next, the C-terminal AAX peptide is cleaved by Rce1. Finally, the newly C-terminal farnesyl cysteine is methyl esterified by Icmt [2]. CAAX processing produces relatively weak membrane affinity and is insufficient to properly localize Ras to the plasma membrane [3]. A second signal immediately upstream of the CAAX sequence affords added affinity. In the case of K-Ras4B, the second signal consists of a polybasic sequence that can interact with the negatively charged headgroups of the phospholipids in the inner leaflet of the plasma membrane.

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