Biology Essay

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.

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1. 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)? The percent acrylamide refers to the size of the pores as percent acrylamide increases the size of the pores decreases.

2. Describe the purpose of each loading buffer ingredient added to protein samples for SDS-PAGE analysis (hint- there are 4 ingredients).

3. 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. The benefit of protein ladder is to have a point of reference for the molecular weight found in the wells next to it. I found that well 2 contained the proteins found in both well 3 and well 4 because it matched up with the bands for each of them. So well 2 evidently has 2 proteins thus 2 bands. Well 3 has one protein 1 band but with a high concentration. Well 4 has 1 protein thus 1 band.

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. Lane 3 is the positive control and lane 4 is the negative control. By comparing lane 4 the negative control and lane 2 the purified protein X it is noticeable that the second band in lane 2 is the elution buffer due to the fact that the negative control only contains elution buffer and only forms one band which matches up with the second band in lane 2.

c. Describe the information you can deduce by comparing and contrasting the positive control lane and lane 2 protein bands. By comparing lane 3 the positive control to lane 2 the purified protein X it is noticeable that the first band is the unknown protein X due to the fact that the positive control only contains purified protein X and only forms one band which matches up with the first band in lane 2.

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). Trends that I see are as the molecular weight decreases the distance traveled increases and the Rf values increases. Rf= distance traveled by protein / total distance traveled

4. 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 spin is enough to separate nuclei from a cell lysate while chloroplasts and lysosomes require higher speeds to separate. Relatively Low speed centrifuge spin is enough to separate nuclei from a cell lysate because the molecular weight difference of nuclei and the cell lysate is very large. Higher Centrifuge speeds are required to separate chloroplasts and lysosomes because they have a small difference in molecular weight.

5. You need to 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
a.i. (V1)(5 mg/mL)=(200uL)( 1mg/mL ) = 40 uL of stock Alpha-tubulin solution and 160 uL of water Has enough stock solution

b. TGFbeta: you have 300 uL at 2.4 mg/mL
b.i. (V1)(2.4 mg/mL)=(200uL)( 1mg/mL )= 83.33 uL of stock TGFbeta solution and 116.67 uL of water Has enough stock solution

c. Albumin: you have 150 uL at 1 mg/mL
c.i. (V1)(1 mg/mL)=(200uL)( 1mg/mL )= 200uL of stock Albumin solution and 0 uL of water There is not enough stock

d. ConA: you have 100 uL at 1.5 mg/mL
d.i. (V1)(1.5 mg/mL)=(200uL)( 1mg/mL )= 133.34 uL of stock ConA solution and 66.66 uL of water There is not enough stock

6. 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? (1L)(10X)=(?)(1X) = 10L 1X buffer made with 1 L 10X and 9L water

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