Star Team

Hi guys, after talking about Biosafety for my 1st 2 postings, today I am going to talk about my MP work flow:Identification and Quantitation of Stenotrophomonas maltophilia periplasmic proteins using 2 Dimensional Gel Electrophoresis and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (2D Gel-MALDI)

For my project, Stenotrophomonas maltophilia (S. maltophilia) was the bacteria used. It is a gram negative bacteria thus it has a relatively large periplasmic space(periplasm) as compared to gram positive bacteria.

Work flow:
S. maltophilia was streaked on the nurtient agar plate and incubated in a 37ºC incubator for 24 hours. After which all colonies will be scraped off the agar plate and reusepened in 5mL of PBS and the cell density is taken. 5X107 cells will then be inoculated into each LB broth and grown at 28ºC and 37ºC in 20mL LB- broth for 18 hours. Cells will be subjected to washing using PBS for the purpose of removing dead cells and proteins that are released due to cell lysis.
Picture taken from: http://rds.yahoo.com/_ylt=A9ibyiCO_gZHbVkAowyjzbkF/SIG=1300lqsrd/EXP=1191727118/**http://www.cofc.edu/~delliss/virtuallabbook/StreakPlates/StreakExamples.html
This is the procedure for cell washing:
1. Take out the cells from the incubator and spin at 3000g for 20 minutes at 10ºC
2. Discard the supernatant into a waste bottle
3. Pipette 10mL of PBS
4. Pipette well and vortex at low speed to resuspend the cells
5. Spin down centrifuge tubes at 3000g for 20 minutes at 10ºC
6. Repeat step 2 to step 5 twice
7. Resuspend the cells in 10mL of PBS
8. Pipette well and vortex at low speed to resuspend the cell pellet

Density of the live cells will then be determined. 6 eppendorf tubes cotaining each
10 to the power of 10 cells will be subjected to extraction using chloroform


This is the procedure for chloroform shock:
1. Spin down cells at 3000g for 20 minutes at 10oC
2. Resuspend pellet in remaining medium by brief vortexing
3. Add 20uL of chloroform to each eppenforf tube
4. Incubate at room temperature for 15 minutes
5. Combine the 6 eppendorf tubes together
6. Add 400uL of 0.1M Tris-HCl to the combined eppendorf tubes
7. Spin at 6000g for 20 minutes
8. Pipette out supernatant in a new eppendorf tube
9. Spin at 14000g for 10 minutes
10. Pipette supernatant in a new eppendorf tubes

Once periplasmic proteins are extracted, microcon purification is done. The purpose of microcon purification is to further concentrate the protein samples


Micron picture. Taken from: http://www.life.sci.qut.edu.au/epping/LSB607OLT/607concentrators.html



Then Bradford assay will be done to give a estimation of the protien concnetration
Taken from: http://www.biocompare.com/review/926/Quick-Start(tm)-Bradford-Protein-Assay-Kits-From-Bio-Rad.html


Once the protien concentration is determined, 2D gel electrophoresis will be done. Basically protiens will be 1st separated according to their pI by isolecetric focusing (IEF) after which according to their molecular weight (for information on how 2D gel electrophoresis is done, refer to Jia xin's blog on Aug 4, 2007)


BIO-RAD PROTEAN IEF CELL used for IEF (picture taken with permission)



BIO-RAD Criterion Dodeca™ Cell used to separate the protein sample according to molecular weight (picture taken with permission)


Once the proteins are separated, the gel is stained with SYPRO RUBY stain for 16 hours and scanned with PharosFX™ Plus Molecular Imager (for information pls refer to MIng Boon's 17th Aug posting)


This is how a SYPRO RUBY stained gel looks like ( picture taken with permission)



This is the PharosFX™ Plus Molecular Imager which I used ( picture taken with permission)



Once the gel is sacnned, quantitation will be done using a software program:PDQuest. PDQuest will allow us to quantitate the gel spots (for this research project, we want to quantify protein spots present in 28ºC and 37ºC with 2-folds) After Quantitation, gel spots will be slected, cut and automatic process by Xcise ( I won't dicsuss about how Xcise works, it was discussed in Ming Boon's posting on 07/07/07)


Xcise machine from Shimadzu Biotech, proteome systems, Japan (Picture taken with permission)


After processing by Xcise, the protein will be spotted on the MALDI plate and subjected to MALDI analysis. The picture below is the MALDI machine used

(picture taken with permission by my supervisor)

end of story=).......
...feel free to ask any question

Eugene Wong
TGO2