TY - JOUR
T1 - High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.
AU - Schrum, Adam G.
AU - Gil, Diana
AU - Dopfer, Elaine P.
AU - Wiest, David L.
AU - Turka, Laurence A.
AU - Schamel, Wolfgang W.A.
AU - Palmer, E.
PY - 2007/6/23
Y1 - 2007/6/23
N2 - Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase-fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.
AB - Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase-fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.
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U2 - 10.1126/stke.3892007pl2
DO - 10.1126/stke.3892007pl2
M3 - Article
C2 - 17551170
AN - SCOPUS:34250684995
SN - 1937-9145
VL - 2007
SP - pl2
JO - Science's STKE : signal transduction knowledge environment
JF - Science's STKE : signal transduction knowledge environment
IS - 389
ER -