Questions FISH PAGE/Western Blot
1. Pls. explain how you extracted protein out of Fish muscle cells. What buffer was used in this process?
2. When you used the Laemelli Sample buffer, there was SDS (a detergent in it). Why was the SDS necessary for running your gel? Why was it necessary for the transfer to the nitrocellulose membrane?
3. When you ran your SDS-Polyacrylamide gel, you ran it using current to move your proteins into the gel and to separate them. Why does the electrical current allow for movement of your proteins through the gel? When running the gel, what properties allow for separation of proteins into distinct bands?
4. Take a picture of your coomassie stained gel, and affix it to the back of the worksheet. Also, calculate the Rf values based on your gel for the protein standards, and draw a standard curve with log [Mol. Wt.] on the Y-axis and on the X-axis plot Rf. You will be given the molecular weights of each band in the standard. To calculate the Rf, take the distance each band migrated from the top of the gel, and divide it by the distance the dye front ran from the top of the gel
5. Using your coomassie stained gel, first calculate the Rf of the bands in the actin and myosin standard lane. Then, using the Rf and your standard curve from question 1, calculate the molecular weight of your bands. Also do this for each fish sample, calculate the molecular weight of two bands.
6. Count the number of bands in each profile, and note the number of bands for each sample in the space below. Do the profiles for each sample have the same number of bands?
7. Which two fish samples have the most similar protein profiles? Why do you think this is the case? Which bands (by molecular weight) are similar between these samples?
8. Which two fish samples have the least similar protein profiles? Why do you think this is the case? Which bands (by molecular weight are different between these samples?
9. Use the evolutionary tree on the back of this worksheet. Do your answers in questions 2 and 3 match the information in the evolutionary tree? Explain your answer to this question in detail.
10. For studying the evolutionary similarities and differences, why is it advantageous to use a coomassie stained gel, instead of doing a western blot?
11. Why is it important when you do the transfer that the gel stay in complete contact with the membrane (no air bubbles)? Also, describe how the transfer works and why the proteins move from the gel to the membrane? How do you know the gel transferred successfully?
9. What protein did we probe for when did the western blot protocol in this laboratory? Explain the steps behind our visualization of this protein, starting with a blank blot.
10. What does the primary antibody recognize and what does the secondary antibody recognize? Which antibody has HRP conjugated to it? What was the function of the HRP substrate?
11. For the protein we probed, was it present in each sample? For your fish samples that we ran that have this protein, is it the same molecular weight throughout. If not, list the samples that contain this protein from smallest to largest. Also, estimate the molecular weight of this protein for each sample.