1	Muscle Biology: A Research Internship Rebecca Johnson October 30, 2003
2	Location Animal Sciences Lab The Ohio State University, 126 Vivian Hall
3	Fellow Researchers Dr. Macdonald Wick, Primary Investigator (P.I.) Chip Pretzman, Research Associate Joe Sawdy, Graduate Student and my Supervisor Scott Updike, Graduate Student John Mark Reddish, Graduate Student
4	Fellow Researchers con’t. Sarah Graham, Graduate Student Grace Key, Work-study Student Frank Cihla, Visiting Research Associate
5	Objective Identify proteins that correlate with tenderness in beef using SDS-PAGE and Mass Spectrometry Show that there is myosin breakdown postmortem
6	What is SDS-PAGE and how does it work? Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Separates proteins based on molecular weight in response to an electric current
7	Why the SDS? Trying to separate based on size so all molecules must be in same linear form with same charge and travel at the same rate SDS acts as a detergent and causes the proteins to unwind—binds to proteins at a constant rate: 1.4g SDS always binds to 1.0g protein
8	Why the PAGE? The acrylamide gel provides a matrix for the protein molecules to migrate through This is what separates the molecules based on size
9	What’s the deal with sample preparation? Reducing Buffer DTT Breaks disulfide bonds and causes proteins to migrate together
10	No Reducing Buffer
11	The Approach Electrophoresis of myofibril samples on a 10% acrylamide gel Stain with Colloidal-Coomassie blue Cut out selected bands and send to mass spectrometry
12	Running Gels Side-by-Side
13	Gel Development
14	Viewing the Gels
15	The Approach con’t. Western blot to show myosin breakdown Transfer proteins to nitrocellulose membrane using electric current to move molecules toward the anode Bind antibodies to proteins on membrane Develop blot
16	Transferring Proteins
17	Identified Bands
18	Determining the Protein Load
19	Trials…. Lab art leaves us asking, ‘Why??”
20	…and tribulations Broken centrifuge, running out of reagents and supplies, smoking machines---all in a day’s work! Welcome to the Wick Lab!
21	Myosin Breakdown! Proving a point via more “traditional” means Western blot indicated what we knew to be true because of mass spec
22	Mass Spec, Here We Come! Phoretixä 1D Software Calculated molecular weights of selected bands Mass Spectrometry Cut out chosen band Protein sequenced
23	Results from Mass Spec!
24	Any Questions?