Using a technique pioneered by Pelham and Wang and refined by others, the role of substrate stiffness in the uptake of the MK2-inhibitor peptides was investigated. Polyacrylamide gels were chosen as the model substrate for this experiment ZK-36374 Because stiffness can be modulated by changing the percentage of bisacrylamide crosslinker within the system. Additionally, polyacrylamide gels are clear, non-fluorescent, and have the ability to covalently link proteins to the surface. Unlike most other systems, polyacrylamide gels are inert to protein adsorption and cell adhesion; thus, cellular adhesion can be controlled by functionalizing the gels with an extracellular matrix protein. The adhesion of cells to the gel is then solely attributed to cellular binding to the ECM protein. Substrates of different stiffness were prepared on 18 mm circle glass coverslips following a modified protocol from Tse and Engler. A uniform film of sodium hydroxide was formed on the coverslips by evaporation of 600 ml of 0.1 M sodium hydroxide in a 60uC oven. In the case that uniform coverage was not achieved, 600 ml of water was added to the coverslips and evaporated in a 60uC oven. The coverslips were reacted with 200 ml of triethoxysilane for five minutes at room temperature under a nitrogen tent, followed by extensive washing with water. The coverslips were then incubated for 30 minutes at room temperature with 0.5% glutaraldehyde. After allowing the coverslips to air dry, polyacrylamide gels were formed on the coverslips under a nitrogen tent. Glass slides were covered with 200 ml dichlorodimethylsilane for 5�C10 minutes and then washed extensively with water. The mechanical properties of the polyacrylamide gels were characterized using an AR-G2 rheometer with a 20 mm standard steel parallel plate geometry. Polyacrylamide gels were made as described and 250 ml of solution was used with a 770 mm gap. A solvent trap was used for all MCE Chemical ON123300 experiments to minimize evaporation. The gelation properties of the polyacrylamide gels were monitored over 45 minutes using an oscillatory stress of 10 Pa and a frequency of 1 Hz. During gelation, the temperature was held constant at 25uC. Because temperature of polymerization has been shown to affect the storage modulus of polyacrylamide
