In this post, I am going to talk about 2 imagers, PharosFX™ Plus and VersaDoc MP 4000. Both the imagers are from Bio-Rad. I will focus more on PharosFX™ Plus as I will be using it for my MP. After running a 2D gel (refer to Jiaxin’s post) and before identifying the proteins (refer to my previous post and Shahirah’s post), the gel image will be acquired using an imager and analysed with a software.
PharosFX Plus main unit, external laser and computer with software
Sample tray to place the gel
The pictures are taken from http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@1782679805.
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PharosFX+Plus+System
PharosFX™ Plus consists of a scanner, a 532nm internal laser and 7 emission filters. As our experiment would also require wavelengths of 488nm and 632nm, an additional external laser was required. The different lasers will excite the respective fluorescent dyes to fluoresce. The wavelength of the fluorescence emitted will be in a range so emission filters are required to filter out other wavelengths, obtaining the desired wavelength. The scanner will then scan the gel from top to bottom and image is obtained. For different applications, different trays are used. For gels that are less than 3mm thick, the default tray (Sample Tray) is used. For microplates and gels that are thicker than 3mm, Multisample Tray 1 is used. For gels that are sandwiched between glass plates, Multisample Tray 2 is used.
Unlike PharosFX™ Plus, VersaDoc uses camera to acquire gel images. Both imagers have their pros and cons. The use of either imager would depend on the application or the type of stain used.
Application
One application of PharosFX™ Plus is that samples (eg. Protein, DNA) in microplates can be quantitated. However, the Sample Tray must be changed to Multisample Tray 1 in order to place the microplate. One application of VersaDoc is that it can be used to do colony counting. Even so, we had to count the colonies manually because we are not able to bring the bacteria out of the BSL.
Type of stain
For fluorescent-, colorimetric- and isotopic-stained gels, it is recommended to use PharosFX™ Plus because in VersaDoc, there is a certain distance between the camera and the gel. The space in between will affect the intensity of the signal captured. With the use of PharosFX™ Plus, the intensity of the signal is fully captured as the gel is in direct contact with the scanner.
For chemiluminescent and ethidium bromide-stained gels, it is recommended to use VersaDoc because PharosFX™ Plus uses scanning (from top to bottom) to acquire the gel image. By the time the scan reaches the end, the signal at the bottom of the gel would have decreased (rate would depend on the half-life of the compound). If VersaDoc is used, the signal will be captured as a whole without any time difference between different points on the gel.
After acquiring the gel image, a 2D analysis software, PDQuest is used to analyse the proteins quantitatively (up/down-regulation) and qualitatively (absence/presence). Quantitatively, we would detect the difference in the intensity between protein spots that are in the same position on two different gels. Usually, only a more than 2-fold increase or decrease will be considered significant because if the difference is only a little, most probably it might be due to system variation (eg. difference due to variation between different machines used) rather than biological variation (difference due to physiological change or drug treatment). Qualitatively, we would detect protein spots that are present in one gel but absent in another gel.
That's all for this post!
Ming Boon
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3 comments:
Hi mingboon,
Ask u ah, how do you all recognise and compare the protein spots on different gels? Do you use any protein markers for recognition?
Royston Tan
TG01
Royston,
The proteins are compared using PDQuest, the software that i had mentioned. Usually, we will run triplicate gels. As we have to compare the proteins between 2 bacteria strains, we will have a total of 6 gels. Using the software, a master gel will be created. A master gel is a virtual gel created by selecting a gel from the 6 gels that has the best quality. This selection can be done by the software or by the user. Protein spots that are found on other gels but not the gel that is selected to be the master gel can be added to the master gel. The 6 gels will then be compared against the master gel. Then you will be able to analyse the proteins qualitatively, where you can see the spots that are present in some gels but not the other gels. For quantitatively, you can select spots that differ in the intensity and use a function in the software to quantify the difference in concentration. Yes we use protein markers but not for recognition. As protein markers have a known molecular weight and concentration, we can use that as a basis to determine the Mw and concentration of the proteins. For the recognition of proteins, it is done by the MALDI, where the proteins are sequenced and identified. Sorry for the lengthy answer. Hope you understand=]
Ming Boon
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