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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

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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

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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 L 50 mM acetic acid to a concentration of 0.5 g/L 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 L 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 L 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

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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 L of H 2 O to make 50 mM DTT stock solution Add 2 L DTT stock to 50 L 0.5 mg/mL AP, making 2 mM DTT in the reaction vessel 3 Incubate the sample in the dark at 50C for an hour to reduce AP Add 25 L of 0.5 g/ 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 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

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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 L of the stock into 100 L of 5 mM Tris-HCl, creating a 0.135 g/L AP solution Prepare three samples of varying protein concentrations with Loading Buffer to run through SDS-PAGE 24 L AP solution with 8 L Loading Buffer for 3.24 g AP/Well 18 L AP solution with 6 L Loading Buffer for 2.43 g AP/Well 12 L AP solution with 4 L Loading Buffer for 1.62 g AP/Well Loading Buffer contains 1 part 4X stacking gel buffer, 1.8 part 10% SDS, 0.2 part -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

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Ratio Trials of Sigma Trypsin Add 12 L of 2.5 mg/mL AP to 200 L 5 mM Tris-HCl, pH 8.0 to create 0.135 g/L AP solution 4 Reconstitute lyophilized Sigma Trypsin in 20 L of 1 M HCl to generate 1 g/L 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 L 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 L 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

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

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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

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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

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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

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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

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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.