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Delivery of bioactive, gel‐isolated proteins into live cells
Author(s) -
Taylor Jennifer E.,
FernandezPatron Carlos
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390171
Subject(s) - gel electrophoresis , polyacrylamide gel electrophoresis , sodium dodecyl sulfate , antibody , biochemistry , membrane protein , intracellular , microbiology and biotechnology , chemistry , immunoprecipitation , biology , membrane , immunology , enzyme , gene
Abstract The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein‐protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep‐1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep‐1 effectively delivers proteins in solution; but Pep‐1 is not suitable for delivering sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) isolated proteins because Pep‐1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS‐PAGE‐isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X‐100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS‐PAGE. Following electrophoresis, the antibody bands were detected by zinc‐imidazole reverse staining, excised, in‐gel refolded with Triton X‐100, and eluted in detergent‐free phosphate‐buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof‐of‐principle for the delivery of gel‐isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein‐protein interactions using gel‐isolated protein.