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10). a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells. == Introduction == Traditional methods for expressing proteins in living cells using gene transfer are often labor intensive, time consuming and can result in formation of inactive or even cytotoxic products (Washbourne and McAllister, 2002). Direct protein transfection eliminates Acetyl-Calpastatin (184-210) (human) the time required for transcription and translation associated with conventional DNA transfection, allowing for more immediate analysis of protein function. It offers a useful tool for studying protein function in living cells and for screening peptide drugs or therapeutic proteins. However, direct delivery of proteins into a large number of cells is typically limited by poor permeability and the potential for denaturation of active proteins during the process. A number of peptides, termed cell-penetrating peptides (CPPs), or protein-transduction domains (PTDs), have been reported to have cell-penetrating properties. These include HIV-1 TAT domain (Fawell et al., 1994;Frankel and Pabo, 1988;Green and Loewenstein, 1988), the third helix of the homeodomain ofDrosophilaAntennapedia (Antp) (Derossi et al., 1994;Lindgren et al., 2000), the herpes-simplex-virus-1 NA-binding protein VP22 (Elliott and OHare, 1997;Phelan et al., 1998), as well as synthetic peptides such as transportan (Pooga et al., 1998), Pep-1 (Morris et al., 2001), YTA2 (Myrberg et al., 2007) and POD (Johnson et al., 2007). These methods have shown high transfection efficienciesin vitroandin vivoand have been used successfully in a variety of cell lines. However many of these methods suffer from some of the same limitations as gene transfection: the methods are labor-intensive and typically require crosslinking between peptides and cargos that can affect function. For Antp and transportan, the proteins or peptides have to be covalently linked to the PTD by chemical reactions (Pooga et al., 1998;Prochiantz, 1996). TAT is either attached directly to proteins by chemical crosslinking or by purifying TAT-containing fusion proteins from bacterial expression vectors. Bacterially expressed proteins lack post-translational modifications and may require denaturation of insoluble protein aggregates, two factors that could alter normal protein function (Nagahara et al., 1998;Schwarze et al., 1999). The VP22 approach is indirect, in that the target cells are transfected with an expression vector containing the cDNA coding Acetyl-Calpastatin (184-210) (human) for the protein fused to the C terminus of MYH10 VP22 (Elliott and OHare, 1997). In contrast to the PTDs and other CPPs, Pep-1 is a short, amphipathic peptide carrier that can deliver peptides and proteins into living cells in a biologically active form without the need for chemical crosslinking or construction of an expression vector (Morris et al., 2001). Commercially available as Chariot (Active Motif, Carlsbad, CA, USA), Pep-1 forms a non-covalent complex with cargo proteins that stabilizes and protects them from Acetyl-Calpastatin (184-210) (human) degradation and facilitates their passage through the plasma membrane. Upon internalization, the complex dissociates, releasing the transfected peptides or proteins into the cytoplasm. Because Pep-1 delivery bypasses transcription and translation processes, the time from transfection to analysis can be as little as 2 hours (Morris et al., 2001), making Pep-1 an ideal tool for protein studies. The Pep-1 reagent has been usedin vitroto deliver antibodies and enzymes into multiple cell types, including osteoblasts (Selim et al., 2003), pheochromocytoma (PC12) (Jiang et al., 2004), Madin-Darby canine kidney (MDCK) and human breast carcinoma MCF7 (Remacle et al., 2005). Pep-1 has also been usedin vivoto deliver caspase-3 (Aoshiba et al., 2003) and cAMP-dependent.