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Winthrop Initiative for STEM Educators 2011 WISE Summer Internship Chemistry Amy Moore, Destinee Johnson, and Derion Reid.

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Presentation on theme: "Winthrop Initiative for STEM Educators 2011 WISE Summer Internship Chemistry Amy Moore, Destinee Johnson, and Derion Reid."— Presentation transcript:

1 Winthrop Initiative for STEM Educators 2011 WISE Summer Internship Chemistry Amy Moore, Destinee Johnson, and Derion Reid

2 Introduction to Experiment

3 Content Research with Mentor TEV protease- Enzyme that clips at known sequence: ENLYFQG/S TEV protease MBP domain N-197 protein Link that includes TEV protease’s clip site The whole protein includes 3 parts, in the middle between the two pieces of protein domain is where TEV protease finds the clip site Clip Site ENLYFQG/S

4 The experiment will place a constant amount of protein together with varying amounts of TEV protease to determine most efficient combination Stable protein amount in each combination Variation in TEV protease amounts in each combination

5 Experiment Preparation

6 Uncleaved Protein MBP and N197 The mass of the uncut protein is approx. 60 kDa (exactly kDa) Gel Electrophoresis confirming presence of protein

7 Post Cleavage Uncut MBP N197 Illustrates all three portions of the protein and confirms cleavage is occurring N197 MBP Uncut

8 Gel of TEV Cleavage Uncut MBP N197 Confirms data from Mass Spec

9 Absorbance of Protein with Standard Protein Assay ●.7757 Final concentration determined for protein = using Spectroscopy How much is there of me(Protein)? Need to know concentrations to find the right ratio of protein to enzyme.

10 Final Experiment

11 Time Dependent Concentration Controlled 1 hour2 hours 3 hours 1 hour2 hours3 hours 1 hour 2 hours 3 hours High Low Electrophoresis gel used to show the relationship between the concentration and time. Medium No TEV protease

12 Concentration Dependent Controlled 0.25 mg/mL Concentration dependent This is a three hours digestion comparing the high concentration of TEV protease to the low concentration of TEV protease mg/mL


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