2. Goals  To find the ideal conditions to perform limited proteolysis  Most efficient trypsin:AP ratio  Buffer solution that optimizes trypsin activity  To determine how the reducing agent DTT affects the structural stability of AP

3. Materials and Methods: An Overview  First, observe proteolysis of Alkaline Phosphatase by Promega Trypsin Gold  Second, trypsinize alkaline phosphatase with and without the reducing agent DTT  Third, optimize AP gel electrophoresis conditions  Fourth, determine the most efficient ratio of Sigma Trypsin to Alkaline Phosphatase for digestion

4. Proteolysis with Trypsin Gold Proteolysis with Trypsin Gold  Take AP, initially at concentration of 1.47 mg/mL, and dilute to 0.5 mg/mL in a 200 mM Tris-HCl buffer, pH 7.4  Bring up lyophilized Trypsin Gold in 200 µL 50 mM acetic acid to a concentration of 0.5 µg/µL 1  Incubate equal parts Trypsin Gold and AP at 37 °C for digestion  This ensured a 1:1 weight ratio of Trypsin:AP  Extract 15 µL samples at 45 minutes and 90 minutes to monitor digestion  Quench digestion for each sample by boiling in water for 2 minutes and subsequently adding 8 µL of loading buffer, boiling again for 5 minutes 2  Run each sample, as well as undigested AP, in a precast 12% acrylamide gel at 200V for ~30 minutes  Stain with Coommassie Blue overnight and destain with 10/10 methanol/acetic acid solution

5. Digestion with DTTDigestion with DTT  Using the same procedure as previously, generate trypsin and AP solutions  Prepare DTT solution for reduction of AP  Mix 0.161g DTT into 500 μ L of H 2 O to make 50 mM DTT stock solution  Add 2 μ L DTT stock to 50 μ L 0.5 mg/mL AP, making 2 mM DTT in the reaction vessel 3  Incubate the sample in the dark at 50°C for an hour to reduce AP  Add 25 μ L of 0.5 μ g/ μ L trypsin to the reduced sample and an equal volume of the same concentration unreduced AP and incubate at 37 °C 2  Also incubate a sample of trypsin for 120 minutes  Remove 10 μ L samples from each reaction vessel at 5, 15, 30, 45, 60, 90, and 120 minutes  Use the same technique as before to quench the digestion  Pour two 15% acrylamide gel, one for each reaction condition  Load each sample, as well as a molecular ladder, undigested AP, and trypsin, running the gels at 160 V for ~45 minutes  Stain and destain as previously described

6. Optimize Electrophoresis Conditions  Generate new stock solution of alkaline phosphatase from lyophilized Sigma AP  Bring up in 1mL 5 mM Tris-HCl to a final protein concentration of 4.5 mg/mL  Dilute 6 µL of the stock into 100 µL of 5 mM Tris-HCl, creating a 0.135 µg/µL AP solution  Prepare three samples of varying protein concentrations with Loading Buffer to run through SDS-PAGE  24 µL AP solution with 8 µL Loading Buffer for 3.24 µg AP/Well  18 µL AP solution with 6 µL Loading Buffer for 2.43 µg AP/Well  12 µL AP solution with 4 µL Loading Buffer for 1.62 µg AP/Well  Loading Buffer contains 1 part 4X stacking gel buffer, 1.8 part 10% SDS, 0.2 part β -mercaptoethanol, 2 part glycerol, and a dash bromophenol blue  Run a precast, 12% resolving gel at 160 volts until AP bands become discernible (20 minutes)  Stain and destain as before

7. Ratio Trials of Sigma Trypsin  Add 12 µL of 2.5 mg/mL AP to 200 µL 5 mM Tris-HCl, pH 8.0 to create 0.135 µg/µL AP solution 4  Reconstitute lyophilized Sigma Trypsin in 20 µL of 1 M HCl to generate 1 µg/µL Trypsin 5  Dilute Trypsin for 1:2, 1:10, and 1:50 Trypsin:AP ratios by weight for digestion  Incubate each sample at 37 °C, removing 10 µL aliquots from each at 5, 30, 60, 105, 150 minutes past initial incubation  Trypsin at each ratio should be incubated to monitor any self digestion, only being removed at 150 minutes for analysis  Quench digestion for each sample by boiling in water for 2 minutes and subsequently adding 3.3 µL of loading buffer, boiling again for 5 minutes 2  Run one precast 12% (160 V for roughly 40 minutes) for each ratio, with wells for a ladder, each time interval, undigested AP, and Trypsin  Stain and destain

8. Day 1 Gel 1:1 Undigested AP T = 45 min T = 90 min

9. Day 3 Gel DTT No DTT M M AP 5 1530 45 60 90 120 T 5 10 15 30 45 60 90 120 T

10. Day 4 Gel 1:2 1:50 1:10 AP M5 30 60 105 150 T AP M5 30 60 105 150 T AP M5 30 60 105 150 T

11. Conclusions  From the results acquired, a 1:50 trypsin:AP ratio, by weight, yielded the most efficient proteolysis in a 2.5 hour time window  Literature research and practical application show a 5mM Tris-HCl at pH 8.0 buffer works for digestion  Use low concentrations of AP and Trypsin so that the trypsin can be heavily diluted into Tris-HCl, maintaining a higher pH than the HCl it is brought up in

12. Ideas for Future ResearchIdeas for Future Research  Allow limited proteolysis to take place for a longer period of time, such as 12-24 hours  Extract bands with good resolution and analyze samples via mass spectrometry  Find which domains are intact after limited proteolysis and  Repeat trials with DTT and use other reducing agents known to affect AP  Use mass spectrometry to compare the intact domains both with and without reducing agents

13. References 1.Promega Corporation (2009) Technical Bulletin Trypsin Gold, Mass Spectrometry Grade, pp 1-3. 2.Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K. (1977) Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and Analysis by Gel Electrophoresis. The Journal of Biological Chemistry 242, No. 3, pp 1102-1006. 3.Sigma Aldrich Corporation (2011) Product Information DL- Dithiothreitol, pp 1. 4.Akitama, Y., Ogura, T., Ito K. (1994) Involvement of FtsH in Protein Assembly into and through the Membrane. I Mutations That Reduce Retention Efficiency of a Cytoplasmic Reporter. The Journal of Biological Chemsitry 269, No. 7, pp 5518-5224. 5.Sigma Aldrich Corporation (2011) Enzyme Explorer Trypsin, pp 1-3.