Sunday, October 28, 2007

Lab Techniques

Trichloroacetic acid(TCA) is one of the many organic solvents used in protein precipitation and purification. It works by disrupting the protein conformation thus exposing more hydrophobic areas, which reduces solubility of protein. In this way, the proteins will be precipitated.

We are using TCA to extract secretory proteins from our bacteria (S.maltophilia) and these are the steps involved.

Day 1 (Streaking)
1. Streak the strain on a LB Agar plate
2. Incubate plate for 24 hours at 37°C

Day 2 (1st Inoculation)
1. Inoculate an isolated colony into 20ml of LB broth
2. Incubate tube for 24 hours at 28
(Remark: The tube is incubated at 28
°C instead of 37°C as most of the strains grow better at about 30°C. We'll like to the bacteria to proliferate so there will be enough cells for 2nd inoculation and for extraction)

Day 3 (2nd Inoculation)
1. Pellet cells at
3000xg for 20 minutes at 10°C
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 20mL of PBS
6. Take OD600 reading in triplicates with 10X dilution
Calculate the volume withdrawal equivalent to 5.0X107 cells
8. Inoculate the calculated volume each into 4 tubes of 20ml LB broth
9. Incubate tubes at 37
°C for 16 hours

Day 4 (TCA Precipitation)
1. Pellet cells at 3000xg for 20 minutes at 10°C
2. Suck up the supernatant (which contains the secretory proteins) with a syringe
3. Filter 18 ml of supernatant through 0.45um filter to remove contaminants (e.g. cell debris) into teflon tubes.
4. Add equal volume of chilled TCA into each tubes
5. Incubate the tubes -20
°C freezer for an hour
6. Invert tubes every 10 minutes interval
7. Spin tubes at 16 000xg, 4°C for an hour
8. Decant the supernatant (beware of dislogded protein pellet)
9. Wash the protein pellets with 250ml of pre-chilled acetone
10. Transfer all protein pellets into a 1.5ml mircofuge
11. Centrifuge at 16 000xg at 4°C for an hour
12. Aspirate supernatant and air dry
13. Resuspend pellet with appropriate amount of rehydration buffer
Acetone is used to wash the cell pellet as TCA is also an organic solvent, like dissolves like so the TCA can be removed. Also,chilled acetone is preferred as low temperature allows the protein to stay in ppt form. When room temp acetone is used, protein pellet turns sticky and thus stick onto pipette tip and tube leading to loss of protein sample. Furthermore, the addition of organic solvent causes heat evolution, care should be taken to keep the temperature low.

Afterwhich, we'll do a protein quantitation to determine the concentration and run a 2D electrophoresis (refer to my first entry), followed by analysis of spots and excision of chosen spots(refer to Ming boon's entry). Finally, to run these peptides in MALDI TOF/TOF (refer to Shahirah's entry) to identfy these peptides. Scroll down if you want to read more about the principles of using these equipments.
That's all! It's the last week that we needa blog! YEAH. Hope you understand and learn something from my posting. See you people in school!

Tang Jiaxin (TG01)


VASTYJ said...

Hi Jia Xin,

How will the seperation of the proteins in electrophoresis be affected if the cell pellet is not washed with acetone? ( ie what is the significance of removing the TCA with acetone)

Interesting post! =)

Loh Sharon, tg 01

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

Hi Sharon,

As mentioned in the blog entry, the TCA changes the conformation of the protein by changing the acidity of the environment, thus changing the isoelectric point (pI) of the protein. Hence, if the TCA is not being removed, it will affect the outcome of the result. In addition, during focusing, the TCA present might also affect the ramping of voltage to the desired 8000V, which is crucial for good focusing in this experiment.