pH 8.6 Tris Partitioning Experiment Gehrke Lab Will Bryant Procedure 1. To do this, 7.

5% (wt/wt) PEG gels were prepared and dried. a. .75g of PEG was added to 9.25g DI water, and 0.013g of Irgacure b. This was mixed for 20min and then loaded into molds c. These molds were cooked at 312nm in a UV-Crosslinker for 50 minutes and then let sit overnight d. They were removed from the molds and placed in DI water with the exception of 3 gels which are weighed immediately and are then leached separately e. The water was changed 3 times over the course of a couple days f. The gels were dried out in a dessicator for 24hrs. 2. Three gels are to be weighed, placed in separate 1.5 mL eppendorf tube, and then soaked in 1.2 mL of each: pH 5.9 Tris buffer solution, buffer with .22M KCl salt, buffer with .22M Bu4NF, a buffer solution, KCl, and 12% (wt/wt) 40,000MW Dextran monomer, or , a buffer solution, Bu4NF, and 12% (wt/wt) 40,000MW Dextran monomer. There are then 3 gels in each solution per experiment and 3 gels are used to obtain the dry weight post leaching. 3. After soaking for 24hrs., the gels are removed and weighed. The gels are then to be moved into the same 1.2mL of solution as before with the addition of 1 mg/mL Ovalbumin. 4. Once the gels are equilibrated (soaked for about 24hrs.) with soln. the gels are removed. Then the gels are placed in their respective buffers with no salt, dextran, or protein. Wet weights of all gels will be taken. 5. The UV-Vis Spectrophotometer is used on the remaining protein solutions to measure the concentration after the gels are removed. A calibration curve of concentrations within the linear range is developed from standards for each solution type. 6. Once the gels, have equilibrated with their buffer solutions, the gels are removed again and the gels are weighed. 7. The leftover buffer solutions are then measured for protein concentration as before. Summary of Data to be collected: y y y y y y y y Mold weights from 1d Dry weights after leaching for % reaction from 2 Initial dry weight for all gels from 2 Wet weight after soaking in protein-less solution for all gels from 3 Wet weight after soaking in protein solution for all gels from 4 Abs. of protein solutions after soak from 5 Final wet weights after leaching from 6 Abs. of buffer solutions after leaching from 7

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