HELLO! =D
If you're wondering what we the in-house students are doing for SIP. NO NO.. not preparing reagents and stock checks! For our group, our assigned task is to be the "supervisors" of the DRP (Differential Research Programs) students!
Johanna and I will be taking one of two groups who are under Dr. Quek's project. This project involves extraction of periplasmic proteins using chloroform (CHCl3) shock, of an isolate of Stenotrophomonas malitophilia which is incubated at two different temperature 28°C (environmental) and 37°C(human body temp.). The differences in protein expression will be analyzed and compared. Experiment will be repeated on other isolates too. This is also the continuation of what Jo and I have been doing for our last DRP.
Outline of Workflow/Protocol
Day 1 (Streaking)
1. Streak the different isolates on a well divided LB Agar plate
2. Incuabte plates for 24 hours at 37°C
Day 2 (Inoculation)
1. All colonies of each isolates is scrapped off and mix in 5mL PBS
2. Cell density (Opitcal Density 600) taken in triplicates to determine number of cell present
3. Volume of inoculum equivalent to 5.0X107 is inoculated into 2 tubes
4. One tube incubate at 28°C and the other at 37°C for 16 hours 250 rpm
Day 3 (Extraction)
1. After 16 hours, the tubes are spin down at 3000xg for 20 minutes at 10°C
If you're wondering what we the in-house students are doing for SIP. NO NO.. not preparing reagents and stock checks! For our group, our assigned task is to be the "supervisors" of the DRP (Differential Research Programs) students!
Johanna and I will be taking one of two groups who are under Dr. Quek's project. This project involves extraction of periplasmic proteins using chloroform (CHCl3) shock, of an isolate of Stenotrophomonas malitophilia which is incubated at two different temperature 28°C (environmental) and 37°C(human body temp.). The differences in protein expression will be analyzed and compared. Experiment will be repeated on other isolates too. This is also the continuation of what Jo and I have been doing for our last DRP.
Outline of Workflow/Protocol
Day 1 (Streaking)
1. Streak the different isolates on a well divided LB Agar plate
2. Incuabte plates for 24 hours at 37°C
Day 2 (Inoculation)
1. All colonies of each isolates is scrapped off and mix in 5mL PBS
2. Cell density (Opitcal Density 600) taken in triplicates to determine number of cell present
3. Volume of inoculum equivalent to 5.0X107 is inoculated into 2 tubes
4. One tube incubate at 28°C and the other at 37°C for 16 hours 250 rpm
Day 3 (Extraction)
1. After 16 hours, the tubes are spin down at 3000xg for 20 minutes at 10°C
2. Decant the supernatant and resuspend the cells in 10mL of PBS
3. Spin down the tubes at 3000xg for 20 minutes at 10°C
4. Repeat steps 2 and 3.
5. Decant the supernatant and resuspend the cells in 10mL of PBS
6. Take OD600 reading in triplicates with 10X dilution
7. Calculate the volume withdrawal equivalent to 1010 number of cells
8. Pipette the calculated volume into 1 microfuge tube
9. Spin down cells at 3000xg for 20 minutes at 10°C
10. Decant supernatant
11. Resuspend pellet in remaining medium by brief vortexing
12. Add 20mL of chloroform to each tube
13. Incubate at room temperature for 15 mintues
14. Add 200uL of 0.1M Tris-HCl to tubes to stop reaction
15. Spin tubes at 6000xg for 20 minutes at 10°C
16. Collect the supernatant to a new microfuge tube
17. Spin the supernatant tubes at 14000xg for 10 minutes at 10°C
- This is to ensure that only the periplasmic proteins are present in the supernatant as cell debris will be pellet down
18. The supernatant is pipetted out into a new microfuge tube
Example of how to calculate 5.0X107 worth of cells
Average OD600 Reading = 1.70
Desired number of cells = 5.0X107
1.00 OD = 1.4X107 cfu/mL
Volume of inoculum = (Desired no. of cell) / (Average OD600 X 1.4X107) X1000
= (5.0X107) / (1.70 X 1.4x107)
= 21.0uL
After extraction, protein concentration will be determined by Bradford assay. The samples will be run on 1-D gel (separation by MW) and stained with either Coomassive Blue or Silver stain. Protein bands will be analyse and compare to identify the difference in protein expression of an isolate incubated at 28°C and 37°C.
Post Stained 1D Gel
Adapted from: http://www.raytest.de/bio_imaging/applications/gel_documentation/applications_geldoc1.jpg
As this is a project that lasts for only 3 weeks, the job load is lesser and simpler.
That's all. Have great day!
Tang Jiaxin TGo1
0503257H
3 comments:
Heyy,
What are periplasmic proteins? On the staining part, in what situations will u use Coomassie Blue or Silver staining? Think u left out the destaining step..
Thanks!
Kent
HeY
Which stain (sliver or coomassie blue) is more sensitive in detecting proteins? Thanks =)
Eugene Wong
TG02
HELLO!
To Eugene:
Silver stain is at least 50 times more sensitive than Coomassie blue. Silver stain is sensitive to 1ng.
To Kent:
Periplasmic space refers to the space between the outer membrane and the plasma membrane of gram-negative bacteria. Thus, periplasmic protein is the proteins present at that area. An example will be superoxide dismutase.
Silver staining method is rather labourious. As it includes steps like sensitizing, developing, staining and destaining. Many reagents will need to be prepared. As for Coomassie blue staining, it takes 16 hours to stain and then destain in MilliQ wather thrice for 20 minutes, lesser steps are needed.
But of course, the sensitivity of silver stain is higher than that of Coomassie blue.
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