Amicon ultracel membrane

Profilin's ability to bind PtdIns(4,5)P₂ micelles was assayed by microfiltration (Haarer et al., 1993 (link); Lambrechts et al., 1997 (link)). PtdIns(4,5)P₂ (Sigma-Aldrich) micelles were prepared at a concentration of 1 mg/ml in water by water bath sonication for 5 min at room temperature. A range of PtdIns(4,5)P₂ micelle concentrations was incubated with 2.5 μM profilin for 2 h at 4ºC. The samples were then transferred to Amicon Ultracel membranes (Millipore, Billerica, MA) with a molecular weight cutoff of 30,000 Da. The samples were centrifuged through the membranes for 1 min at 2000 rpm. The flowthrough was collected, run on a 15% SDS–PAGE gel, and stained with Coomassie Blue. The intensity of the profilin bands was measured by an Odyssey infrared imager (LI-COR).

Bcl-xL siRNA, PLL (MW = 30,000–70,000 g/mol), and diethyl pyrocarbonate (DEPC)-treated water were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bare AuNPs (size: 40 nm) were purchased from BB International (Cardiff, UK), Amersham Cy5.5 Mono NHS Ester was from GE Healthcare (Buckinghamshire, UK), Amicon Ultracel membranes (10 kDa) were from Millipore (Billerica, MA, USA), and sodium HA (MW = 100–150 kDa) was from Lifecore Biomedical (Chaska, MN, USA). KLA peptide was synthesized as described.²⁷