By Isabelle Mus-Veteau Ph.D. (auth.), Isabelle Mus-Veteau (eds.)
Membrane proteins play a key function in several pathologies akin to melanoma, cystic fibrosis, epilepsy, hyperinsulinism, and Alzheimer’s ailment, but reports on those and different issues are hampered by way of an absence of knowledge concerning the proteins concerned. In Heterologous Expression of Membrane Proteins: equipment and Protocols, specialist researchers supply an summary of different heterologous expression structures to be had to provide those proteins for structural research, supporting us to appreciate how those proteins functionality, and also, how their services could be converted via drug remedy. Chapters learn membrane protein solubilization, purification, and instability in resolution, and examine the ideas that helped to figure out the constitution of the 1st heterologously expressed mammalian membrane proteins. Composed within the hugely winning Methods in Molecular Biology™ sequence layout, every one bankruptcy encompasses a short creation, step by step equipment, a listing of important fabrics, and a Notes part which stocks tips about troubleshooting and fending off identified pitfalls.
Far-reaching and leading edge, Heterologous Expression of Membrane Proteins: equipment and Protocols, is a vital consultant for amateur and skilled researchers alike, delivering unique tools for current expression platforms along state-of-the-art suggestions for destiny use within the box.
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Additional resources for Heterologous Expression of Membrane Proteins: Methods and Protocols
Do not make any changes in the primer extensions required for LIC. Genomic DNA is easily compromised by repetitive freeze/ thawing, vortexing, or vigorous pipetting and thus requires careful handling; storage in sterile tubes at 4°C is preferred. Combine PCRs for targets of similar length and template type (plasmid or genomic) as much as possible instead of performing a PCR for each gene separately. Touchdown PCR ensures a more specific product formation (42), and as a range of annealing temperatures is used, it is a good alternative to optimizing the reaction conditions for each target on an individual basis.
Store at −20°C. 3. Vector Backbone Exchange 1. 5 mL of SfiI (5 U; see Note 19). 2. Digest the plasmids for 1 h at 50°C in a PCR machine with heated lid to avoid excessive evaporation. Subsequently, inactivate SfiI by heating for 20 min at 80°C (see Note 19). 3. 5 U; see Note 20). Incubate for 1 h at room temperature and inactivate the ligase in a PCR machine by heating for 20 min at 65°C. Store the sample at −20°C. 4. Electrotrans formation of L. lactis 1. Thaw electrocompetent L. lactis NZ9000 (see Note 21) on ice.
Analyze the protein content of the fractions in the different elution peaks by SDS-PAGE on a 12% SDS-polyacrylamide gel. 8% SDS, pH 8 buffer (see Note 22). Recover the dialyzed protein and, eventually, concentrate so that the final volume does not exceed 3–4 mL (see Note 23). 8% SDS, pH 8 buffer. 2 mL/mL; 1-mL fractions). Analyze the protein content of the fractions in the different elution peaks by SDS-PAGE on a 15% SDS-polyacrylamide gel. Discard the aggregated fractions and pour the fractions containing the nonaggregated receptor protein.