Lipid solution (A) of COATSOME® SS-20/3A-P04 (NOF America corporation, White Plains, NY): cholesterol (Merck, Darmstadt, Germany): SUNBRIGHT GM-020 (NOF America corporation, White Plains, NY) = 70:30:3 (mol) was prepared in 90% t-BuOH. siRNA solution (B) was prepared by mixing 100 μl of human SOCS1 siRNA (NM_003745) or contr. siRNA (SIC003) (Sigma-Aldrich, Taufkirchen, Germany) aqueous solution (133 μg/100 μl) and 50 μl 20 mM malate buffer (pH 4.00, 30 mM NaCl). For enclosure of tetanus light chain toxin (List Biological Laboratories, Inc., California, USA) 5 μg/100 μl was added instead of siRNA. While vortexing 400 μl of the lipid solution, siRNA solution (or TeTxLC) was gradually added dropwise and the mixed solution was taken in a 1 ml syringe (C). Then, 225 μl of (C) was injected into 1 ml malate buffer, with vigorous stirring (D). Thereafter, the mixture was further stepwise dialysed with PBS. Adding 2 ml PBS was followed by centrifugation for 1 h at 1,400 rpm in a concentrator plus (Eppendorf, Wiesloch, Germany). Procedure was repeated four times. Final siRNA concentration 66.5 μg/ml. In the experiments 50 μl/ml and 100 μl/ml L-siRNA, 40 μl/ml L- TeTxLC was used.
Product #650A, Tetanus Toxin Light Chain, Recombinant, has been discontinued. Large quantities of Product #650A may be obtained through Bulk Requests and Customer Orders.