Individual Blog Post #2

Surprises and challenges that our group encountered during the experiment were smearing that occurred in both of our gels. Smearing occurred in our gels because in both trials, we may have cut up the muscle proteins from our six species in different sizes, and many of them were cut large in size. Furthermore, in the second gel, we came across a challenge with transferring the buffer from the fliptop microtubes to the screw top microtubes. There was not enough buffer to transfer in some of the flip top microtubes, which occurred in the tubes that contained muscle protein that was cut up very largely. We added more buffer to these microtubes, but it still may have caused errors in the second gel and may have resulted in the smearing in the gel. Also, a lot of bubbling occurred when transferring the buffer from the flip top to the screw top microtubes, which may have resulted in less buffer settling in the screw top microtube and may have caused smearing in our gel.

Advice and solutions that I would give to other groups is to cut the muscle proteins from the six species in relatively small sizes so that there will be less chance of smearing in the gel. This will also ensure that there is enough buffer to transfer from the flip top to the screw top microtubes because the protein will not be so large as to absorb the buffer, which was assumed to have happened in our experiment. Furthermore, I would advise groups to carefully transfer the buffer from the flip top to the screw top microtubes to reduce bubbling. This will ensure that enough buffer is transferred to the screw to microtubes, and will reduce errors such as smearing in the gels.

Research Project Blog Assignment #2

We designed our project to compare muscle protein bands in skeletal muscle and and cardiac muscle from four different organisms. We were afraid that our first gel would be blurry or not run evenly because we loaded samples in all ten lanes but the gel turned out well. Our challenge was related to how long it took us to be measure our bands in lab. The bands had begun to fade and we were concerned that we were unable to measure all of them. We had the intention to run a second gel and measure the bands sooner but our second gel was much blurrier and did not run as evenly as our first gel even though we thought we repeated the process the same way as the first time.

In our error analysis we will likely include that we should have kept the amount of specimen we used from each organism more consistent by weighting them.

Research Project Individual Blog Assignment #2

By doing this project so far, one thing really confuses us is the result part. We did use the same procedure for both lab,however, after running the second gel, we find out its much different than the first one. The second gel actually shows up that it has more bands than the first one and appears that the second one is more indistinct. We made a decision to use the data from the first gel, but we don't know how will this affect our results. In our project, we have protein bands from two types of muscle, heart and breast from several different species. The second thing confuses us is, even though those protein bands are from different parts and species, but they look similar to each other. Except for the outgroup, there is not much significant differences between those bands. We didn't expect this when doing our hypothesis. And the members in our group have measured the distance between each bands, but how do we know which one we should use to avoid the errors.

Individual Blog Post #2: Group Project Update

Jennifer Chin

24 Feb. 2017

BIOL&212 AA

     A surprise our group encountered was how similar the octopus protein bands were to the mussel protein bands instead of the similarity between mussel and scallop. A challenge our group encountered were the smearing of the gels and the cutting of our proteins. In both trials, we tried to keep the proteins in similar small-sized cuts, to avoid major smearings. We did not weigh the amount we cut because of time consumption. Another challenge during our second trial we faced was not proportioning the amount of buffer each flip top microtube dispensed. Some flip top microtubes had more buffers than others, causing the tubes with less buffer to be absorbed in the protein, and resulting being unable to transfer the liquids to the screw top microtubes.

     In the group's error analysis, we will discuss the different sizes of the muscle proteins of our samples and how it made smearings in our gel. Also, the amount of buffer used in each test tube during the second trial. The buffer bubbled a lot when pipetting into the flip top microtubes, after resulting at the end of the room temperature incubation, some flip top microtubes did not have any liquids to transfer to the screw top microtubes. We used some extra buffer to get our results, which still weren't the best. Both errors messed up our gels, causing smears. Another error could be the amount of time our Actin and Myosin standards were in the incubation chamber. The standards were not in for five minutes, but instead for three minutes. This could have made our gel from trial 1 lack certain bands to distinguish the actin and myosin for our other proteins.

Aaron Oberstadt

The majority of our group's problems match those of others in the class. We decided to repeat the fish protein lab with various mollusks. This proved to be difficult to exactly replicate in our second gel as the mussel samples had all died. Like Asmah mentioned, we had significant bubble production when transferring Laemelli buffer. Any major issues with bubbles or liquid that would not settle to the bottom of a tube, we found, could be solved with a centrifuge. Finally, on our second gel, a large number of our protein bands were extremely faint, which made reading it very difficult.

These issues, among others, will be addressed in our error analysis. Lacking a second sample of mussel is potentially a major cause of error in our final results, and we could not have prepared for that. We did not maintain consistency in who transferred the protein samples from their respective tubes to the polyacrylamide gel, which could be a source of error. The second gel ran for 5 minutes longer than the first gel, potentially explaining the faintness of its protein bands. Finally, our inability to measure bands that should exist but are too faint to accurately see can also contribute to error in our results.

Research Project Blog Assignment #2

Nitesh Chetry

2/24/2017

Biol& 212 AA

Research Project - Individual Blog Assignment #2 

      Looking at the data from the gels obtained in lab, one of the biggest surprises we encountered was how different the pig was to sheep and cow. We expected the sheep and the cow to be very similar to each other because of the similar digestive tracks, as explained in my Blog Assignment #1. Also, we expected the pig to be the next distant relative to the sheep and cow because of similar hooves. Instead the bear, a non-domesticated animal seemed to share more protein bands with the sheep and cow than the pig shared with the sheep and cow. This can be seen on both our gels, from the two separate lab days, so we know that this likely didn't occur due to an error. Overall, I was very surprised to see very small differences between the protein bands of all our animals.

     I am curious to know how to other groups dealt with the any denaturation or protein band streaks that would make it difficult to identify certain bands. For my group, we noticed small streaks in around the same spot for all of the animal proteins. We tried to improve this in our gel two by using 9.5 micro liters instead of 10 micro liters. However, the same problem occurred, and we found similar streaks in our second gel. I wonder if any groups, who observed streaking in their first gel, were able to make any significant changes that fixed the problem for their second gel. Also, I'm curious to see how common this small amount of streaking was overall, for all of the groups.

Research Project Blog #2

My group's goal is to see how well the muscle proteins within bear, caribou, deer, elk, moose, and chicken legs differentiate from one another. Our hypothesis is that, while the proteins of all four species of the deer family will be fairly similar to each other, deer and elk will be most closely related to each other, and caribou and moose will be the closest related to each other as well. However, we found the surprising result of bears having very similar proteins in their muscle tissues as the other mammals! I suppose it's not TOO surprising, since bears are also mammals but the results are actually very identical. (see below) As a matter of fact, all the sampled animals have relatively similar proteins in their muscles, with chicken being the one who holds the most difference.

Lane components:

1. Empty
2. Kaleidoscope standard
3. Elk
4. Moose
5. Bear
6. Chicken leg
7. Caribou
8. Deer
9. Actin & Myosin standard
10. Empty

Figure 1. Result of the first gel electrophoresis.

Figure 2. Result of the second gel electrophoresis.

Due to this, we're faced with greater challenge of distinguishing the differences between each muscle tissue. The bands on the second gel are also smeared in many places, most likely due to too much muscle tissue in the sample or poor pipetting technique, making it even more difficult to read the results. There could also be contaminants on the razors that would interfere with our data, all of which will be discussed in the error analysis section of our project. I'd like to advise other groups to choose a variety of species for more interesting results, and to avoid cutting too much of the muscle tissues when preparing your samples for gel electrophoresis. Less is more!

Individual Blog Post #2

The only challenge that our group encountered with is that the gels from both trials came out looking smeared. On the first trial, some of our bands came out smeared. We believed this was due to having different sizes of fish muscles from the different organisms.

On the second run, we tried to cut all the fish samples as similar as we could, however some of our bands still came out smeared. In this trial, we definitely had more errors compared to the first trial we ran. One error we encountered with is when we were putting the buffer into the flip tops, it formed bubbles so then when we added the fish muscle, we realized we weren’t able to extract enough protein so we ended up putting more buffer solution into some of the flip tops. This could also explain the smearing on the bands. Another error in this trial is that our fish muscles could have also been different sizes like it was in the first trial. At first we thought of weighing all the fish samples to make sure they were about the same weight before adding the buffer but then we decided against it because we thought that it was unnecessary and it would take up too much time. My question is did any groups weigh the fish muscle before putting it into the buffer solution? If so, did it make a difference weighing it vs not weighing it?