MCT techniques and a the machines stuff..

Hmm..okie..it's my turn again after Nat..this time I'm going to talk more about my MP.

Basically, my MP is about the protein analysis of lung cancer cells that are treated with herb using LC-MS/MS. It mainly involves MCT techniques and chemical engineering stuff such as the machines used. Prior to using the machines, I have to seed cells and here's where all the aseptic techniques and cell counting comes in. Using a hemocytometer, the cells are counted and the concentration of the cells are calculated. This had been elaborated in Najib's entry, so take a look if u guys don't know how to alright! A cell densitiy of 7million cells are seeded and 3 of the culture dishes are treated with 2mg/ml of herb extract after a 24 hr incubation time. Why 2mg/ml then? well, this concentration had been verified by a senior that it is the optimal concentration that will cause a lowest cell viability. An incubation time is required to allow the cells to adhere to the surface of the culture dish and also to proliferate somemore! After that, the cells are killed!! M-PER is added to allow the cell lysis so that protein extraction can be done using a cell scraper. The supernatant is then taken out and stored at -80degrees after a centrifuge step.

Now comes the part that is rather foreign to us, as it's rather chem eng based. 2D-LC is run! Before this, the proteins are trypsin digested and iTRAQ labeled. iTRAQ reagents will label proteins based on the different mass, namely 114,115,116,117. These reagents binds to the peptides (i.e. proteins after the trypsin digestion) at the N terminal of the lysine chain. This labeling will allow the quantification of the different proteins identified. In addition, it allows up to 4 samples to be mixed and run together, therefore lowering the time needed for the run to be carried out! This step then makes my life easier, because i will know if the protein is being up or down regulated based on the iTRAQ ratio that is given.

Ok! back to 2D-LC. 2D-LC is actually a fractionation step to fractionate the peptides samples. This involves 2 column modes, column 1 and column 2 mode and 2 different pumps are also involved, the quaternary pump and the capillary pump. In column 1 mode, the quat pump is the busy one, it pumps the sample that is injected into the Strong Cation Exchange (SCX) column then to the enrichment column and lastly to the waste. The peptides will then be stuck at the SCX column. In column 2 mode, the cap pump becomes the busy one. It pumps the peptides that is stuck at the enrichment column into the RP column. Erm, as some of the peptides will be still stuck at the SCX column, salt of different molarity is run. These salts help to elute the stuck peptides that is at the SCX column into the enrichment column. The irritating thing about this machine is that it lacks a auto fractionator, which means that I'm the fractionator. The eluted samples are collected at a 2mins interval. Which means that at every 2min, I have to change the tube to a new eppendorf tube. Imagine sitting at the lab for straight 4 hrs!! Can't leave the lab at all. Sickening rite!

Now here comes MALDI. MALDI helps in the protein detection and the protein will then be identified by the MASCOT system. I will not be elaborating on MALDI as Sharhirah had already talked about.. so guys..refer there! :)

Okie..thats about all..Just a brief idea on what my MP is actually about and the stuff involve.. If there's any questions, just post them k..I'll answer them asap!

Have Fun people! Back to school soon..haha..

Charmaine
0503186I