Sunday, September 2, 2007

Lab Techniques

CyDye Fluor in DIGE

Hi guys! Hope all of you are doing well in the 10th week of your attachment, 10 more weeks to go so hang in there all right. For those who are enjoying your work, appreciate every week cause all good things must come to an end :p

For this blog entry, I will be sharing with you guys about CyDye used in DIGE. DIGE stands for DIfferential Gel Electrophoresis. It is a technique used to detect and identify proteins involving 2D-gel electrophoresis (scroll down to Jiaxin’s entry for more info). It allows up to three different protein samples to be separated on the same gel. This ability is termed multiplexing. Each protein sample is labeled with one type of dye i.e. CyDye in this case.

Now what is CyDye then? Basically it is a cyanine, fluorescent dye used to label the proteins. There are different forms of CyDye depending on the sample you are dealing with. For example there is a set of CyDye to label scarce samples. Since the sample is limited, a lot of dye is added and bind to the proteins at certain amino acid residues such that they become saturated with the dye. The saturation will help in the detection of the presence of the proteins after 2D- gel electrophoresis. In general, CyDye is excited lasers at specific wavelengths and emits a signal of a narrow wavelength range. There are three CyDyes used in most experiments; Cy2, Cy3 and Cy5. Cy2 is excited by a laser at a wavelength of 488nm whereas Cy3 is excited at 532nm and Cy5 at 635nm.

For my group’s experiments, we will most probably use one of the three dyes i.e. Cy3. We are only using one as we intending to test if this method of labeling is suitable for the bacterium that we are working on i.e. Stenotrophomonas maltophilia. At the moment we are trying out a protocol to extract only the exposed cell surface proteins of this gram-negative bacterium and we are using this dye to label the surface membrane proteins.

As mentioned earlier that there are various forms of cyanine dyes depending on the experiment. The Cy3 that we are using is a minimal dye. This dye has an ester group on it. It will bind only to the epsilon amino group of lysine found on proteins to form an amide linkage. Most proteins have a number of lysine residues on them thus these proteins will be labeled with the cyanine dye. It is termed a minimal dye as it is limiting in the reaction between the proteins and the dye. Only a small amount of the dye will be added to ensure that only 1-2 % of the available lysine is labeled with the dye. This is so as during identification of the proteins using mass spectrometry like MALDI (refer to my previous post), the proteins will be cut using trypsin. Trypsin cleaves at lysine. Thus, we use minimal labeling such that it will not affect the mass spectrometry results. Once the proteins are labeled, the sample will be subjected to gel electrophoresis. For my experiment, the sample will be run on 1D-gel where the separation of proteins is based solely on its molecular weight.

That will be all for my post. Take care and say hi to week 11!

Posted by: Shahirah Bibi


VASTYJ said...

hey.. hi.. juz wondering.. since cyanide is involved.. are there a nid for any extra protection besides double gloves? Thanks..

Jia Hao

first6weeks said...

Hi Shahirah.

I would like to know briefly what is the difference between using 1D and 2D gel.

Desmond Heng

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

To Desmond
Hi there, well in 1D we separate the proteins solely on its molecular weight while in 2D we separate it based on its pI(the pH of the protein)and then its molecular weight.
For more info, you could look at jiaxin's entry. hoped i have answered your question.

To Jia Hao
We need to ensure that there isn't any naked flame during one step where we reconstitue the dye with DMF (dimethyformamide) as this chemical is highly flammable. yup that's pretty much about it.

I apologize to the both of you for the delayed response :p