Saturday, August 4, 2007

Lab Techniques

Hi everybody! I'm attached to a research center which all of you are VERY familar with. =D My MP and SIP are done together with Ming Boon, Johanna & Shahirah. Our research revolves around proteomic (protein-related studies) work and the bacteria that we are looking at is Stenotrophomonas maltophilia. It is a Gram-negative bacillus which causes nosocomial infections. We are working on 2 different strains, the environmental (from soil) and clinical (from patient infected with S.maltophilia). Hopefully, we will be able to identify the difference in protein composition between the two strains, which makes the clinical strain infectious.

As you know, a cell is made up of many different kind of proteins; secretory, cell membrane, cytoplasmic and etc. So, it would probably take years to study and analyze all of them! We were given a visable goal which is to look into only 2 types of protein; secretory and exposed cell membrane proteins. These 2 types of proteins are selected as a microbe usually attaches to and infects a cell through secreting proteins or sticking onto it through the exposed cell proteins. At this point of time, we are still analyzing the secretory proteins.

The secretory proteins alone consist of many different proteins. In order to analyze individual proteins, they first have to be separated. For our research we're using the 2-Dimensional Electrophoresis approach.

First Dimension Separation : Isoelectric Focusing (IEF)

Proteins are amphoteric molecules such that they can carry either positive, negative or zero net charge depending on the pH of their local environment. For every protein, there is a specific pH at which its net charge turns zero. This is its pI. pI refers to isoelectric point and is defined as the pH at which a protein will not migrate in an electric field and is determined by the charges it carries.

The proteins are placed in a medium with a pH gradient. When electric field is applied, they will move towards the electrode with opposite charge (unlike pole attracts). During migration through the pH gradient, proteins will either gain or lose protons leading to decrease in net charge and mobility. Ultimately, the protein will reach a point where the pH gradient is equal to it's pI. Being uncharged, migration would stop. In any case where a protein were to diffuse to a pH region lower than its pI, it will be protonated and forced back towards the cathode by the electric field. On the contrary, if it diffuses to a region of pH gradient greater than its pI, the protein will be negatively charged and move towards the anode. Through this way, focused spots are obtained.

P.S. The circles represents the proteins and the number
within it is its pI

Adapted from:

For our project, the pH gradient used is a strip of acrylamide matrix gel that has pH gradient incorporated covalantly making it immobolized even under electric field. These strips will have to be rehydrated with rehydration buffer and protein samples, together with the buffer, the proteins to be separated will be absorb into the gel strip. The gel strip used is called IPG strip.

Second Dimension Separation : SDS PAGE (Molecular Weight)

The pH gradient strip is then placed in the well of the acrylamide gel for 2nd dimension separation.

The proteins on the pH gradient gel migrates down, separation based on molecular weight.

Reminder: The numbers refers to protein's pI

The black spots indicates the stained and separated proteins

Adapted from:

The protein spots will be excised with Xcise (scroll down to view Ming Boon's post) and analyzed with MALDI (scroll down to view Shahirah's post).

THAT'S ALL for my post! =D

Tang Jiaxin


BloodBank.MedMic.Haematology said...


Is there any disadvantages of using Isoelectric Focusing (IEF)? If don't use IEF but only SDS PAGE, will the results be affected? Thanks

Ci Liang

royal physicians said...

Hello =)

Er, what does IPG stands for?
And why does the strip has to be rehydrated?


Chen Kangting

J.A.M.M.Y.S said...

For Ci Liang:

Some of the disadvantages of IEF:
1. The IEF gels are expensive,
2. The electrophoresis takes about 16 hours, time consuming,
3. Some highly hydrophobic proteins are lost during IEF as they are not solubilize.
4. A small amount of protein might be precipitated due the overlapping (see diagram in blog entry).

If we only use SDS PAGE method the proteins are only separated by molecular weight. This is a 1 Dimension separation What we like to have a more refined separation thus we use the 2Dimension separation.

For KangTing:

IPG stands for Immobilized pH Gradient. The strip is sold in dehydrated form thus it has to be rehydrated to open up the pores of the gel allowing the proteins to enter.

Hope it answers your questions and cleared your doubt. =D

take care!