During the beginning of the semester, I managed my CHO-cells for a week and prepared for a luciferase assay on the 3rd of February by counting my cells. Using the hemocytometer I was able to get a good number of cells to plate into wells and prepare for transfection the next day. On the next day, I transfected all of the TORCA mutants along with their wildtypes, a negative control and a tax control. On February 5th, I was able to do my luciferase assay. Once I completed the assay, I put the data in an excel data sheet and generate a graph in order to analyze my results. The results were not ideal since our TORCA mutants did not express more gene expression than their wildtypes. I discussed this with my professor and was told to repeat the experiment again.
On the 2nd of March I counted my cells and only had half of my last experiment. However, the cells looked like they were in good condition so I was told to experiment on them. I transfected successfully the day after and the day after that, I did my luciferase assay. Once again the results for this assay was not ideal. We saw that the Tax gene expression was less than the negative control which is not normal. From there, we decided that my next luciferase assay would be to test the Tax samples to determine if they have been contaminated. After finding out what the results are for our tax samples, we would be able to proceed with the project. I’ve learned over and over again how every little factor could affect your experiments. With enough patience and persistence however, we would be able to get ideal results.
The results that I got from my immunofluoresence experiment did not provide us with much information since there was a lot of background. We used a new antibody in hopes that it would provide us with better results than our last immunofluoresence where we used a anti-goat antibody. This time we used a v5 antibody and received a lot of background. We will attempt to redo the experiment but instead of keeping my research at a halt, we also did another luciferase assay to check up on the mutants to make sure that they are fine.
The results I got from my luciferase assay were also strange. Ideally, our mutants (TORC1-3 A and D) should have much more gene expression than their wildtypes (TORC1-3). Our results did show that the TORC mutants had more gene expression than their wildtypes but it did not look significantly higher. Also, our tax + torc wildtypes had more gene expression than the torc mutants with tax. Ideally, the torc mutants with tax should be more than the torc wildtypes. Some questions that came up while analyzing these results are: why are torc mutants + tax not the highest gene expression and what can we address to fix it?, should we attempt to retry the immunofluoresence with the v5 antibody or stick with the anti-goat antibody?, should we stop all other experiments and attempt to make new mutants? We plan to analyze all of our experiment results and try to come up with a conclusion for our next step.
My upcoming research will involve immunofluorescence of HTLV TORC proteins. Last year I worked with luciferase assays in CHO cells and this summer I worked on immunofluorescence of 293T cells. For this year’s research, I will be working on immunofluorescence of CHO cells. After obtaining data for immunofluorescence of CHO cells, I would be able to continue doing luciferase assays in CHO cells. The immunofluorescence of CHO cells helps confirm the localization of the mutants I created during my first research project.
For this project, I expect to get immunofluorescence pictures showing clearly the localizations of each of the TORC proteins (1-3) and their mutants A and D. I hope to learn more about immunofluorescence since it is still somewhat new to me. In doing immunofluorescence, I would be maintaining a cell line, splitting cells, transfecting and then washing the cells and attaching them onto cover slips. I would then take allow the cover slips to dry and analyze them under a microscope.
On my last blog, we tested TORC3 proteins with the rabbit antibody. Unfortunately we did not get the ideal results. The results showed mainly background and we were unable to get clear shots of the cells. This may be due to the antibody so we wish to test another antibody.
This time we decided to test all of the TORC proteins with a new antibody. We inquired a tax antibody (mouse – 1315) and tested it along with the His mouse antibody on all of the TORC proteins. We had 24 samples and had 4 controls that had no transfection. We recently just finished the immunofluorescence and are waiting for the slides to dry for the results. A question we had from our first experiment was: are the cells fluorescing their ideal colors under the microscope. Since they were not, we were able to conclude that our primary antibody was the problem. Since then we were able to advance and attempt to retest everything with another primary antibody.
This project was challenging because staining cells can be difficult. There are so many factors that can mess up the cover slips. My first day attempting immunofluorescence took me 6 hours. However, since then I was able to cut down the time to 4 hours. From this project, I was able to learn more hands-on techniques, and learned more about antibodies in general. This project was a new experience for me and I hope to continue it during the school year.
Our experiment is to do immunofluorescence on HTLV TORC proteins. The reason we are doing immunofluorescence now is to check on my results received from my luciferase assays. The immunofluorescence is used to check the co-localization of the TORC proteins. This allows us to verify whether our TORC mutants are nuclear or cytoplasmic. As mentioned in my previous blogs, our TORCA mutants ideally should be dephosphorylated and would move into the nucleus whereas our TORCD mutants ideally should be phosphorylated and would move into the cytoplasm. With the immunofluorescence experiments, we should be able to found out if our mutants are working correctly.
In order to do immunofluorescence, we must first manage a cell line of 293T’s which are human kidney cells. We then have to count and split the cells into plates so that we can evaluate them individually later. The following day, we would have to transfect the proteins into the 293T’s. The day after we would have to fix the cells and then do immunofluorescence. Once the immunofluorescence is done, we are able to view the slides under the microscope to see whether our mutants are cytoplasmic or nuclear.