Week 19- 2D-SDS-PAGE & 2D-Zymography

Hello guys...One more week to go before the end of SIP/MP. In my final post, I shall mention on the last phase of my project, which is the running of 2D-gel electrophoresis. The 2D-gel electrophoresis will be divided into the 2D-SDS-PAGE and 2D-Zymography. For those who are still wondering what zymography is, please refer to Benjamin's entry for more information. Before I continue, let me define these two gel electrophoresis techniques.

2D-SDS-PAGE: It is an electrophoretic technique which uses a SDS-polyacrylamide gel to separate proteins based on molecular weight only (function of SDS molecules).

2D-Zymography: It is an electrophoretic technique which makes use of the same principle of how 2D-SDS-PAGE works and uses a SDS-polyacrylamide gel co-polymerized (in simpler term: combined) with a protein substrate, gelatin. The addition of gelatin into the gel is to detect protease activity, which is our main interest.

Following extraction of periplasmic proteins by chloroform shock (recall from my previous post), we shall screen for the isolate of S. maltophilia where its periplasmic proteases exhibit the highest protease activity. This can be achieved by running the 1D-zymography which is thoroughly elaborated by Benjamin's post. On the 1D-zymogram gel, protease activity will be visualized as clear bands (transparent bands). The S. maltophilia isolate(s) which has the most number and largest clearings will most likely be our isolate of interest and we will culture this isolate for the 2D-SDS-PAGE and 2D-zymography.

The reason why we are detecting these periplasmic protease activities is because these proteases might be potential virulence factors of S. maltophilia which cause diseases in humans. Hence, it is of great significance that we identify these periplasmic proteases and hopefully in the years to come, new vaccines can be created to target against these virulence factors.

The difference between 1D-zymography and 2D-zymography is that in 1D-zymography, proteins are separated based on molecular weight only. However in 2D-zymography, proteins are separated based on 2 dimensions, where the first dimension separation is isoelectric focusing (IEF), where proteins are separated based on their isoelectric points. The second dimension separation is SDS-PAGE, where proteins are separated based on their molecular weights. On the 1D-zymogram gel, proteins will appear as bands. On the other hand on the 2D-zymogram gel, proteins will appear as spots. Periplasmic protease activity will be visualized as clear (transparent) bands on the 1D-zymogram gel as compared to the 2D-zymogram gel where periplasmic protease activity will be visualized as clear spots. Logically speaking, proteins undergoing 2 dimensional separations will be more resolved as comapred to 1 dimensional separation.

In summary...

1D-zymography: 1st dimensional separation- SDS-PAGE
Proteins are separated based on their molecular weights (MW).

2D-zymography: 1st dimensional separation- Isoelectric focusing (IEF)
Proteins are separated based on their isoelectric points (pIs).

2nd dimensional separation- SDS-PAGE
Proteins are separated based on their molecular weights (MW).

In 2D-gel electrophoresis for our research application, it is necessary to run the 2D-SDS-PAGE and 2D-zymography together on the same time. This is because we would want to detect the region of periplasmic protease activity on the 2D-zymogram gel and match that region to that on the 2D-SDS-polyacrylamide gel. If there is protein spots on the 2D-SDS-polyacrylamide gel corresponding to the location of the clearings on the 2D-zymogram gel, it will most likely imply that those protein spots are the periplasmic proteases accounting for the clearings on the 2D-zymogram gel.

Ultimately, the main highlight of our project is to obtain the identity of the periplasmic proteases. However before this can occur, we must first develop and optimize our 2D-zymography protocol, which is the gist of our project and it is still ongoing. Wish us luck and all the best for the last lap of SIP/MP...!!! :)


Name: Tan Han Yang
Class: TG01
Admin no. : 0606190G