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Your lab has determined a section of the Rat Brain is involved in cheese foraging

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Your lab has determined a section of the Rat Brain is involved in cheese foraging. Rats that have injury in that area do not eat or seek cheese snacks. That brain cell region is known as Get The Cheese (GTC). Each question 33 points in value.

Q1) Given your cell biology knowledge of cell-lines, detail a series of experiments to isolate a GTC cell line. Additionally, you must include your control experiments for the above techniques to confirm your experiments and results.  Also include a flow diagram of your techniques, experiments, and controls.

 

Q2) You next turn to GTC protein analysis. You are in search for GTC intracellular specific proteins that lead to the brain function in Rat’s GTC behavior Using your knowledge of protein purifications and 1D and 2D SDS PAGE techniques find specific GTC protein using other brain (OB) cell lines as controls to eliminate common brain specific proteins. Additionally, you must include your control experiments for the above techniques to confirm your experiments.  Also include a flow diagram of your techniques, experiments, and controls.

 

Q3) Using the protein purification techniques, proceed to generate monoclonal and polyclonal antibodies to GTC protein, sequence these GTC specific proteins via Edman degradation, and confirm usefulness of these antibodies by Western Blotting. Additionally, you must include your control experiments for the above techniques to confirm your experiments.  Also include a flow diagram of your techniques, experiments, and controls.

 

Q4) Next, you move to using flow cytometry and FACs (4 quadrants) to repurify GTC cells for primary cell culture testing confirming your results in part Q2 and Q3. Additionally, you use the Antibodies from Part Q3 to observe what intracellular organelles these proteins are in. Confirm by centrifugation, organelle fractionations and Western Blots.

You are expected to apply various centrifugation techniques including Differential/Density Gradient/Rate Zonal/Sedimentation Centrifugation. All your protein/organelle fractions will also be tracked by Spectrophotometric Analysis to track proteins within the subcellular fractions. Confirm your fractionations for purity, amounts of the Golgi/ER/Nuclear etc. membranes and proteins, and prove the fractionations worked.

 

Q5) After the experiments detailed in Question 4 you find the GTC brain cells have strange looking Golgi with 4 stacks instead of 3. Purify the GTC and OB cell specific Golgi organelles, protein fractionate using protein chromatography and HPLC to isolate just the Golgi proteins from both cell lines.

These Golgi proteins will then need to be visualized using 1D and 2D SDS-PAGE looking for differences in GTC vs OB cell lines. Western Blotting using polyclonal and or monoclonal antibodies will confirm protein levels and identity, protein similarity between the 2 cell-lines and protein differences.

1) Detail your experiments for first purifying the Golgi proteins, visualizing the Golgi proteins, and conformational experiments as described above.

2) Include all the needed techniques and control experiments as well

3) Include a flow diagram of your experiments using the techniques and controls.

4) Do the different tissues have different protein levels in their Golgi? Or the same. Discuss this key question!

 

Q6) Your lab now has GTC cell lines correctly labeled and analyzed, useful antibodies, pure GTC and normal brain Golgi fractions as well as a proteomic map of the key GTC Golgi proteins.

Given your knowledge of the Cell Free System first developed by the Classic Dr. William Balch Experiments which included Virus Glycoproteins, Mutant Cell Lines, Invitro Assays, Carbohydrates and Golgi Structure, design a series of experiments to develop a cell free system to test the hypothesis that the Golgi from the GTC and normal brain cell-lines to determine if they can interact and be both donor and acceptor Golgi elements (Cis-Golgi, Medial-Golgi, Extra GTC Golgi Stack and Trans-Golgi).

1) Discuss your experiments to test this hypothesis. You are free to use all techniques will be discussed as well as others if needed,

2) Included the essential controls experiments

3) Outline the series of experiments you use to set-up and demonstrate you have a successful cell-free system