reclip (reversible cross-link immuno-precipitation) an efficient method for interrogation of labile protein complexesreclip(可逆交联immuno-precipitation)一个有效的方法不稳定蛋白复合物的审讯.pdfVIP

ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes 1 2 1 1 1 Andrew L. Smith , David B. Friedman , Huapeng Yu , Robert H. Carnahan , Albert B. Reynolds * 1 Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America Abstract The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure ‘‘ReCLIP’’ (Reversible Cross- Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes. Citation: Smith AL, F