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
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
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


Extract 15 µL samples at 45 minutes and 90 minutes to monitor digestion


This ensured a 1:1 weight ratio of Trypsin:AP
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
Digestion with DTT


Using the same procedure as previously, generate trypsin and AP solutions
Prepare DTT solution for reduction of AP


Add 2 μL DTT stock to 50 μL 0.5 mg/mL AP, making 2 mM DTT in the reaction
vessel 3




Also incubate a sample of trypsin for 120 minutes
Remove 10 μL 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


Incubate the sample in the dark at 50°C for an hour to reduce AP
Add 25 μL of 0.5 μg/μL trypsin to the reduced sample and an equal volume of the
same concentration unreduced AP and incubate at 37 °C 2


Mix 0.161g DTT into 500μL of H2O to make 50 mM DTT stock solution
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
Optimize Electrophoresis
Conditions

Generate new stock solution of alkaline phosphatase from lyophilized Sigma
AP



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






Bring up in 1mL 5 mM Tris-HCl to a final protein concentration of 4.5 mg/mL
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
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
Day 1 Gel
Undigested AP
T = 90 min
T = 45 min
1:1
Day 3 Gel
DTT
No DTT
Day 4 Gel
1:2
1:50
1:10
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
Ideas 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
References
1.
2.
3.
4.
5.
Promega Corporation (2009) Technical Bulletin Trypsin Gold, Mass
Spectrometry Grade, pp 1-3.
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.
Sigma Aldrich Corporation (2011) Product Information DLDithiothreitol, pp 1.
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.
Sigma Aldrich Corporation (2011) Enzyme Explorer Trypsin, pp 1-3.