What i have been doing for LSM2201A: PAGE

Hi guys,

Today I will like to report on what I have done so far for our LSM2201A.

For our last 2 practicals, we have been doing polyacryamide gels electrophoresis(PAGE). What is that? Basically, PAGE is a common method of analysing proteins. There are 2 kinds of PAGE: native and SDS.
Firstly, we have to prepare the polyacylamide gels. Polyacylamide gel is the preferred choice for electrophoresis due to the following factors. It ,depending of how you might want to vary your recipe, can form in a wider range of pore sizes.( for reasons we might want to see later.) and compared to the other common gel medium, agrose, it is more durable and resilient. Usually, most of the time, the gel would be 2-layered discontinuous system: Resolving gel at the bottom and stacking gel at the top. The theory behind this set-up are out of scope for this post but this discontinuous set up promotes better separation of the proteins (resolution) Polyacryamide gels are composed of mainly acylamide monomers and N,N methylene bis acylamide monomers to cross link the chains formed. As a result, we can get a uniform mesh of pores through biomolecules can migrate. This reaction needs radicals to get going so to do just that, we can add ammonium persulphate with N,N,N,N tetramethylethylene diamine to generate free radicals. For those who have some chemistry background, you will be certain to understand this point more better. After we prepare the gel, we loaded our gel into the electrophoresis apparatus provided to us and loaded our protein samples (prepared from our previous experiments) into the wells in our gels using the micropipette. My TA made sure that everyone of us had a go at that as that is an a important skill in modern biology. Sounds difficult, but it becomes not as difficult as it seemed once I and my lab mates had a go at it. =). After we had loaded everything, we switched on the power supply. Once the electrophoresis started, usually It will take some time to perform the process. So we had 1 hour to go for lunch break and got to know my lab mates better =). When the protein reached the other end of the gel, we stopped the process and removed the gel. Very carefully indeed. Now we have a new problem: the protein bands are totally invisible...and how on earth are we going to see them? Usually, the gels undergo staining and de staining with the dye Coomassie Blue, which stains proteins and give blue bands we can see. Staining stains the whole gel blue and de staining removed any dye that have not bound itself to the protein, thus leaving the blue bands.

OK...so you may ask: what is the difference b/w the native and SDS PAGE?

For that the key concept is that electrophoresis is a good way to separate proteins as a result of an applied electrical field. Now this separation is determined by both size and net charge of the proteins. Let's us take two proteins with the same molecular mass and shape. The one with the greater net charge will go faster towards the other side of the gel. Native gel electrophoresis is usually carried out if we want the proteins to remain in their natural shape and conformation. Why would we want to do that? Well, one reason for that would be if we wanted to carry out activity staining. For our experiment, to test for the dehydrogenase activity we used tetrazolium salts which are lightly coloured when oxidised and coloured when reduced.. This stains the bands and allowed us to see in which band has the most enzyme content and activity.

DS stands for sodium dodecyl sulphate. SDS makes a good detergent which can create a good lather: in fact it is found in products like toothpastes, shampoos and other cleaning and hygiene products. Now, SDS binds to the hydrophobic areas of proteins causing them to unfold into polypeptide chains. Molecular mass is now the sole determining factor on the migration rate of the protein. Shape is no longer a factor due to the fact that the native structure are completely unfolded by the SDS so the shapes are the same. Proteins bind to the same number of SDS such that they have the same amount of negative charge. Larger proteins will have slow migration rates than smaller proteins due to the fact that they cannot pass through the pores of the gels as easily as their smaller counterparts. The end product is that on the gel, we can see a series of protein bands arranged in terms of molecular weight. By plotting the ratio of the distance moved by the protein band and the distance moved by the proteins (called the Rf value)against the log of molecular mass (Log transformation to give the straight line) of known proteins standards, we can figure out the mass of an unknown protein. Cool =) However, the method has certain setbacks. One possible one if the protein has post-translation modifications like glycoxylations made to it, it will move anomalously on the gel and give a misleading result.

Just realized the week I have 2 full labs: LSM2201A (the fith practical) and CM1101. Fun!!!

Till next time =)

Chinese New Year

Chinese New Year was an eventful (and a very profitable one)one for me. Having fallen in the midst of recess week, there was plenty of time to celebrate. The pessimistic ppl would probably think that the recess week was desingned to allow the Chinese students to go back to China or something. On the eve on Chinese New Year, my extended family visited my house for reunion dinner. Then I went to River Hongbao with my sister and her boyfriend. It was a beautiful sight to see to all the decorations. The next day, we were off to our paternal grandmother's house in Toa Payoh. On both of these nights, after the visiting was over, we went over to Selagie Road for LAN gaming to play Left for Dead 2. It was my first time playing, and while i succesfully completed the frist campaoign with my sister without 'dying', I didn't quite make it for the rest of the rounds.=( Nethless we had an enjoyable time with her friends fromwork.
A couple of days later, I had my aunt from Australia visiting us. We headed towards Sentosa and ate in Resorts World. She gave me $100, A$100 and US$100 sealed in a big red packey =)=) We tried some of the food and we found the food deliclious. After that, while my aunt decided to gamble and win $500 at the casino, we went to visit Underwater World.

My Underwater World videoclip can be seen here:
http://www.youtube.com/watch?v=lMyHc-DF8gs

I brought the A Guide to Sponges of Singapore at the giftshop. After that we went walking in Vivocity. I brought a packet of assorted chocolates at the Candy Empire. Overall, it was a nice day out for us.Haha. The next day, I went with my nephew and his mum to his party at Hougang. He , before he went to RI, he went to St'Andrews Junior School and his CCA is rugby. That party was their Chinese New Year Reunion party and the party was held at one of theparents house. Netheless, me and my nephew played with the roulette wheel and the poker cards with his friends =)=) And eating all kinds of yummy food. Oh, before i went home, i watched Everton beating Man United 3-1 on cable TV..Drats.=(
The sum total of these outings? 18 red packets and $520 dollars. By my standards, it was a rich pickings this year. Usually, my extended family gives $120 in all.
How do you guys celebrate Chinese New Year?