Weird... Felt like writing yet again the exciting stuffs that happened to me during my RA and master degree period. Learnt a lot of stuff during this period of time. I had chosen to write out the things I learnt as I remembered them.
After the acceptance by my supervisors, I got busy with the preparations of proposal. And of course the application forms were abit of problems as my referee reports got lost even I sent them in end of July by my referees' courtesy. That aside, coming up with a proposal title proved to be a headache. I am glad that my supervisors chose to let me do things on my own just after giving me slightest idea about what is expected. Prof. Sim gave me five (5) snakes to choose upon for my research. They are (i) Malaysian pit viper; (ii) Malayan cobra; (iii) Malayan krait; (iv) king cobra; and (v) Malaysian lance-headed pit viper. I chose Malayan krait as my snake and hand in the proposal for first review.
After both supervisors discussed among themselves regarding my proposal, it was concluded that the snake proposed wasn't the best choice. Malayan cobra seems to be a better choice as the venom composed of three major components: (i) PLA2; (ii) neurotoxin; and (iii) cardiotoxin. In short, this is the perfect snake as the venom has different mode of action. Sufficient for my studies. We have tonnes of the venom in the lab, readily available.
After a few more reviews from both of them, my proposal is completed. This is the period where my Aax2 will be having her convocation. After her convocation, we went back to Sandakan together. Shortly after that my grandma passed, if not mistaken at 7.35am on 5th of October 2007, Friday where it is the last day of PMR. Thank God that I was back home where I am helping out in handling some of the logistic matters. After the burial on the 10th or 11th, my mom and younger brother stayed back a week longer as my bro was having after PMR break and I believed my mom wouldn't want to return home after awhile. I decided to spend my time with them until they leave KL. Thank God that both my supervisors let me do so and it was then that I realised all of them in the lab (except for two Malay lab assistants) are Christians! Amazing! It was then that I finally realized that I am in good hands.
I got along with them very well, except for the PHD student and PRof. Tan as I don't see them that often. Shin Yee was having her annual break and was expecting a child soon. Therefore I supposed the bossy-instructing attitude could be mainly due to the hormonal issue. Prof Tan in the meantime always "sigh" in his office. Thus I keep my distance from him, afraid that I add on to his burden in anyway. Nevertheless, they are pleasant people to get along with by the way they interact with others.
Kak Shikin taught me the first few days to do SDS-PAGE and Western Blotting using Invitrogen Western Blot Kit. The SDS-PAGE kit was quite difficult to master as it always leak. Through many practices and leaving it overnight are the keys to ensure correct setup. I had be repeating SDS-PAGE and Western Blot for about 6 times in about a month. It was then the chromogen from the kit expired and thus the work stopped until now. The blotting stuff hadn't arrive yet tough the chromogens arrived in January. I did also column chromatography during this period of time. The velvet bean extracts was ran through the Con-A Sepharose column to isolate the glycoprotein of interest, which shows off as a peak in elution buffer tube 7 onwards by Bradford assays. Again, Bradford assays were repeated over and over again to finally get a reliable result. I noticed that there are two ways to prepare the Bradford solution.
There are three ingredients needed in order to prepare the Bradford solution:
(i) 100ml of 85% (v/v) phosphoric acid
(ii) 100mg of Coomassie Brilliant Blue G
(iii) 50ml of 95% ethanol
The instructions to prepare them is simple: 100ml of 85% (v/v) phosphoric acid was added to 100mg of Coomassie Brilliant Blue G and dissolved in 50ml 95% ethanol, followed by topping it up to final volume of 1 liter. One of the lab assistant followed this method and the resulting solution is dark blue in colour. I noticed that when we use this method to prepare the BRadford solution, the powder took a very long time to dissolve completely. I wonder whether we should add everything up then only stir to dissolve. The other lab assistant however, mixed the two solution first before adding the powder. In this case, the powder dissolved easier and the resulting solution is more black than blue in colour. The first method of preparation gave fluctuating results whereas the second method gave a more stable and consistent results. I read about Bradford assay and most of the literature confirmed that the first method of preparation to be correct. Then where did it go wrong? Beats me... Given the many factors that may influence the results, the time and incubation time are perhaps the variables.
Imagine this: I have 60 samples to be run using Bradford's assay. Each smple requires three replicates. The BSA standard required 33 test tubes alone! Therefore, in total there were about 33+(60*3)=213 test tubes to be read using the spectrophotometer a day... SEGOII... Each test tube are required to be read three times. Gulp** It took me a whole day to read and finish all the samples. I had to prepare the solution a day ahead and kept them in dark.
After getting used to the techniques and time management, slowly better results came into view. Once I asked my Australian senior regarding the preparation of Bradford Solution, he showed me a literature that I came across before. When I asked him whether he faced any complications in preparing the solution, he answered that he didn't know of any. Guess what? He had been using commercially available Bradford solution all this while and never tried bancuh sendiri. Sweatnya >_<" Another alternative to do Bradford assay is by using 96 well microtiter plate. This seems like a better alternative. In this case, a multichannel pipetter can be used and therefore the inconsistency could be minimised greatly. And not to mention that we can also minimised wastage as both the samples and solutions required are greatly reduced. Thinking that I had found the possible solutions, I proposed this alternative to the lab assistant. However, the answer that came back to me was "we never did this in this lab before. We have always used the cuvetter for this all the while... " >~<"
After PRof. Tan was finally satisfied with the results, we moved on to yet another chapter: Building up immunity of rabbits using the MPE fractionated by column chromatography. Of course, this is yet another chapter of my story...
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