Experiments (Write protocols and perform experiments based on these):

1. Diluting the SDS Buffer Solution
2. SDS-PAGE Experiment

The prelab must include Experimental Protocol, Chemical Table and Equipment Table.

The lab report requires all sections (including prelab sections) to be completed in one document.

1. (Manipulation) Calculate how many microliters of BSA solution you would need to load 50 micrograms of BSA onto the gel. (The concentration of the BSA solution is 1 mg/mL.)
2. (Manipulation) Calculate the volume needed to prepare 500mL of 1X SDS-PAGE running buffer, from a 10X stock solution.
3. (Existing knowledge, research and views) Find and list the molarity of the 10X buffer using the published literature. Include your reference.
4. (Manipulation) Assuming that your known protein of 75kDa molecular weight migrates 2.5cm, identify the molecular weight of a protein that migrates 4 cm. Show all the steps in the calculation of the molecular weight of the protein that migrates 4 cm.
5. (Representation) Take a photo of your gel and use it to draw an image (could be hand-drawn or digitally rendered) that accurately represents your results. Be sure to label which lanes are the ladder/reference, and which are samples.
6. (Interpretation) Identify each protein either with a different color or some other identifier on the above image.
7. (Assumptions and Analysis) Fill in the following table using the observations and data from your experiments.

Write a minimum one-page (12 font, single spaced) discussion on the experiment conducted this week. Address as many questions as possible in each category as fully as possible integrating the collected data, providing explanations for the observed trends, and evaluating whether your original assumptions about the experiment were validated by the results. The assignment will be graded on completeness, clarity of the explanations and the meaningful integration of the collected and calculated data. Correct grammar and appropriate format for the chemical formulae and chemical reactions is expected.

1. (Existing knowledge, research and views) Classify the chemicals used in this experiment as polymers or monomers. Identify the structures of monomers used.
2. (Existing knowledge, research and views) Define buffers and identify the buffer used in this experiment
3. (Existing knowledge, research and views) Define 10X. Provide at least one supported argument for why this unit of concentration is more appropriate for this experiment compared to others.
4. (Existing knowledge, research and views) Describe the SDS buffer solution and explain its role in the SDS-PAGE experiment.
5. (Existing knowledge, research and views) Identify the proteins used in the experiment and describe their major characteristics relevant to the experiment.
6. (Existing knowledge, research and views) Define electrophoresis and present how it was applied in your experiment.
7. (Analysis) Give at least 2 supported arguments for the need for a thin gel in this experiment.
8. (Lab skills) Give a supported argument for using the micropipette to mix your solution components.
9. (Lab skills) Give a supported argument for when it is necessary to change micropipette tips and when it is unnecessary.
10. (Analysis) Give a supported argument for heating the sample to 95 °C for 5 minutes.
11. (Analysis) Describe how the size of a protein affects the migration rate. Relate this to your observations.
12. (Analysis) Discuss how the voltage applied during the run affects the resolution of proteins in SDS-PAGE.
13. (Analysis) Describe how the pH of the buffer affects the protein migration.
14. (Existing knowledge, research and views) Define the Rf value; and describe the properties of the protein that will contribute the most to Rf value.
15. (Existing knowledge, research and views) Describe the concept of staining and destaining in the context of the SDS-PAGE experiment.
16. (Existing knowledge, research and views) Identify the structure and binding properties of the Coomassie dye,
17. (Existing knowledge, research and views) Identify the purpose the Coomassie dye serves in an SDS-PAGE experiment.
18. (Interpretation) Describe the observed protein bands (position and intensity) from your SDS-PAGE gel compared to the molecular weight marker.
19. (Analysis) Use the molecular weight marker to estimate the size of the protein you isolated.
20. (Interpretation) Describe your protein under study as a function of molecular weight and predict how it should appear compared to the known protein.
21. (Analysis) Describe any inconsistencies between the observed and expected results of your SDS-PAGE experiment and provide a supported argument for what effect may be responsible for them. (HINT: you might consider any unexpected bands, smeared bands, degradation, incomplete denaturation in this category.)
22. (Assumptions) Describe at least one assumption that you made in your experiment.
23. (Experiment design) Propose a protocol for investigating a protein that has a molecular weight twice as large as your current protein. Describe the steps that you would keep the same and the ones you would modify.