Due to the week of Nov. 10-14th
You may use the lab manual, pre-lab lectures, and credible internet resources, however you may not use your cell bio lab classmates as a resource. You will most likely see this material again on the Final and I highly encourage you to work individually and seek help from myself or your TA.Plagiarism will result in an automatic zero.
1. [15pt]In the cell bio lab, we use company manufactured gels, however you can make you own polyacrylamide gels. List all of the ingredients found in an SDS-PAGE gel. Which ingredients are responsible for polymerizing the solution? How does the percentage of acrylamide effect the migration of proteins (ex: 4% gel vs. 18% gel)?
2. [8pts]Describe the purpose of each loading buffer ingredient added to protein samples for SDS-PAGE analysis (hint- there are 4 ingredients).
3. [45pts]You purified protein X via affinity chromatography (no diafiltration step performed) and ran an SDS-PAGE gel of the sample with a set of controls. Below is the result of your SDS-PAGE analysis.
1 2 3 4
Figure 1. SDS-PAGE of purified protein X. Lane 1, Protein ladder (in Daltons). Lane 2, purified protein X (affinity chromatography). Lane 3, purified protein X (company manufactured). Lane 4, elution buffer.
a. What is the benefit of a protein ladder/molecular weight marker in an SDS-PAGE gel? Describe what you can learn about the protein bands found in lanes 2, 3 and 4 based on the protein ladder bands. b. Positive and negative controls are absolutely essential to the validity of experimental data. In this gel above, which lanes contain the positive and negative controls? Describe the information you can deduce by comparing and contrasting the negative control and lane 2 protein bands. c. Describe the information you can deduce by comparing and contrasting the positive control lane and lane 2 protein bands. d. You don’t know the molecular weight (MW) of protein X and you are not able to find that information in the scientific literature. The best way to determine the MW of a protein using an SDS-PAGE gel is to use the protein ladder bands to create a Log(MW) vs. Rf graph and calculate the MW from the line of best fit. What is the equation to calculate the Rf of a protein band? Make a table of the Log(MW) and the Rf values for all 5 protein ladder bands. Describe any trends you see in the table values. Sketch a scatter plot of the data (log of MW on the y-axis).
4. [10pts]In lab 9, we will use differential centrifugation to separate cauliflower cell organelles into individual fractions. Describe the basic principles of differential centrifugation. Discuss why a relatively low speed centrifuge spinis enough to separate nuclei from a cell lysate while chloroplasts and lysosomes require a higher speeds to separate.
5. [16 pts] You needto prepare 200 uL of each protein at a concentration of 1mg/mL for an upcoming experiment. Calculate the recipe to make each desired solution at the above concentration and volume. Do you have enough materials to make each dilution? Note: You diluent is water. a. Alpha-tubulin: you have 500 uL at 5 mg/mL
b. TGFbeta: you have 300 uL at 2.4 mg/mL
c. Albumin: you have 150 uL at 1 mg/mL
d. ConA: you have 100 uL at 1.5 mg/mL
6. [6pts]You have 1L of 10X SDS-PAGE Running Buffer and you need to make as much 1X running buffer as possible for the upcoming experiment. How much 1X running buffer can you make using the entire 1L of 10X running buffer? (Must describe what the number means!)
For BONUS POINTS5 pts(applied to HW#3 grade), students must use Excel to plot a log(MW) vs. Rf graph of their gel standards and add a polynomial (2nd order) trendline. Students must then calculate the MW of one protein band in ConA sample lane using the graph. How does that number compare with their visual estimation? Directions for these steps are in the prelab lecture material and on a separate worksheet found in the SDS-PAGE content folder on Learn.