2 replies to this topic

[mreza]

Hi everyone

I use following protocol for TCA precipitation, i can see the protein pellet before centrifugation, but after that it disapears. does anyone know the reason?


Precipitation Protocol:
1. Add TCA to final conc of 20% .
2. Incubate 1 h at -20 c.
3. Spin tube in microcentrifuge at 14K rpm, 10 min.
4. Remove supernatant.
5. Wash pellet with cold acetone.
6. Spin tune in microfuge at 14K rpm, 5min.
7. Repeat steps 4-6 for a total of 2 acetone washes.
8. Dry pellet by placing tube in 95°C heat block for 5-10 min to drive off acetone.

[fishdoc]

mreza, on Jul 22 2009, 03:55 AM, said:

Hi everyone

I use following protocol for TCA precipitation, i can see the protein pellet before centrifugation, but after that it disapears. does anyone know the reason?


Precipitation Protocol:
1. Add TCA to final conc of 20% .
2. Incubate 1 h at -20 c.
3. Spin tube in microcentrifuge at 14K rpm, 10 min.
4. Remove supernatant.
5. Wash pellet with cold acetone.
6. Spin tune in microfuge at 14K rpm, 5min.
7. Repeat steps 4-6 for a total of 2 acetone washes.
8. Dry pellet by placing tube in 95°C heat block for 5-10 min to drive off acetone.

After which centrifugation do you lose the pellet? If it's after the acetone wash, I've had that happen, too. Don't know why it happened, but I thought maybe it was do to some water having gotten into the acetone, so that it was no longer 100% acetone.

I was using the preps for 2D-PAGE analysis, so I skipped the acetone step and resuspended the pellet after TCA precip in water. It didn't actually solubilize in the water, more like a suspended precipitate. I then used the ReadyPrep 2D Cleanup Kit from BioRad, which is basically a fancy acetone precipitation, to clean up the protein sample and resuspend it in rehydration buffer for 2D.

[BIOKMST]

Did you know that TCA will not work if 1) the amount of TCA added is too large and 2) if the amount of protein you're after is under 1ug?  Good Luck!