Experiment Details - Experimentium

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Experiment of SDS-PAGE Details

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Experimental Protocol

Chemical Table

Equipment Table

Data Collection

Data Processing

<ol>
 	<li>(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.)</li>
 	<li>(Manipulation) Calculate the volume needed to prepare 500mL of 1X SDS-PAGE running buffer, from a 10X stock solution.</li>
 	<li>(Existing knowledge, research and views) Find and list the molarity of the 10X buffer using the published literature. Include your reference.</li>
 	<li>(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.</li>
 	<li>(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.</li>
 	<li>(Interpretation) Identify each protein either with a different color or some other identifier on the above image.</li>
 	<li>(Assumptions and Analysis) Fill in the following table using the observations and data from your experiments.</li>
</ol>
<table>
<tbody>
<tr>
<td width="164">Assumptions made</td>
<td width="240">Testing the assumption</td>
<td width="219">If assumptions are wrong ...</td>
</tr>
<tr>
<td width="164">Micropipettes are accurate.</td>
<td width="240"></td>
<td width="219"></td>
</tr>
<tr>
<td width="164">Distilled water is pure.</td>
<td width="240"></td>
<td width="219"></td>
</tr>
<tr>
<td width="164"></td>
<td width="240"></td>
<td width="219"></td>
</tr>
<tr>
<td width="164"></td>
<td width="240"></td>
<td width="219"></td>
</tr>
</tbody>
</table>

Discussion

<p>Write a minimum one-page (12 font, single spaced) discussion on the experiment conducted this week. Address <strong>as many questions as possible in each category</strong> 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. <strong>The assignment will be graded on completeness, clarity of the explanations and the meaningful integration of the collected and calculated data.</strong> Correct grammar and appropriate format for the chemical formulae and chemical reactions is expected.</p>
<ol>
 	<li>(Existing knowledge, research and views) Classify the chemicals used in this experiment as polymers or monomers. Identify the structures of monomers used.</li>
 	<li>(Existing knowledge, research and views) Define buffers and identify the buffer used in this experiment</li>
 	<li>(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.</li>
 	<li>(Existing knowledge, research and views) Describe the SDS buffer solution and explain its role in the SDS-PAGE experiment.</li>
 	<li>(Existing knowledge, research and views) Identify the proteins used in the experiment and describe their major characteristics relevant to the experiment.</li>
 	<li>(Existing knowledge, research and views) Define electrophoresis and present how it was applied in your experiment.</li>
 	<li>(Analysis) Give at least 2 supported arguments for the need for a thin gel in this experiment.</li>
 	<li>(Lab skills) Give a supported argument for using the micropipette to mix your solution components.</li>
 	<li>(Lab skills) Give a supported argument for when it is necessary to change micropipette tips and when it is unnecessary.</li>
 	<li>(Analysis) Give a supported argument for heating the sample to 95 °C for 5 minutes.</li>
 	<li>(Analysis) Describe how the size of a protein affects the migration rate. Relate this to your observations.</li>
 	<li>(Analysis) Discuss how the voltage applied during the run affects the resolution of proteins in SDS-PAGE.</li>
 	<li>(Analysis) Describe how the pH of the buffer affects the protein migration.</li>
 	<li>(Existing knowledge, research and views) Define the Rf value; and describe the properties of the protein that will contribute the most to Rf value.</li>
 	<li>(Existing knowledge, research and views) Describe the concept of staining and destaining in the context of the SDS-PAGE experiment.</li>
 	<li>(Existing knowledge, research and views) Identify the structure and binding properties of the Coomassie dye,</li>
 	<li>(Existing knowledge, research and views) Identify the purpose the Coomassie dye serves in an SDS-PAGE experiment.</li>
 	<li>(Interpretation) Describe the observed protein bands (position and intensity) from your SDS-PAGE gel compared to the molecular weight marker.</li>
 	<li>(Analysis) Use the molecular weight marker to estimate the size of the protein you isolated.</li>
 	<li>(Interpretation) Describe your protein under study as a function of molecular weight and predict how it should appear compared to the known protein.</li>
 	<li>(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.)</li>
 	<li>(Assumptions) Describe at least one assumption that you made in your experiment.</li>
 	<li>(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.</li>
</ol>