Week 18 Attachment Sharing

Alright, this week i'll be sharing more on the analysis, excision and identification of proteins. Also, i'll touch a bit on the DRP group that me and Jiaxin were put in charge of. This weeks blog is more of a reflective entry rather than a factual one.

After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.

Protein spot analysis
After extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.

For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.

Protein spot excision
The protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.

After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.

After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.

DRP students
For our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.

In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.

Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.

Johanna
0503309G
TG02