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
Slide 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
Slide 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 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
Slide 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 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
Slide 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
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
Slide 7
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
Slide 8
Day 1 Gel 1:1 Undigested AP T = 45 min T = 90 min
Slide 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
Slide 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
Slide 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
Slide 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
Slide 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.