Hard to dissolve pelleted protein after TCA precipitation - Urgent!! Please check this out!! (May/08/2007 )
Hi folks,
I am doing IP (w/ a-flag Ab) and then elution with 3x flag peptide. Then to concentrate the IP'd proteins, I am trying TCA precipitation. But someone said that it is really hard to dissolve pelleted protein with 1x sample buffer for SDS-PAGE. Right now I am at step 2 (O/N).
Below is my protocol and does anyone have good idea to dissolve pellet (at step 8)?
Many thanks to all of you!
1. Add 1 volume of TCA stock to 4 volumes of protein sample.
2. Incubate 15 min on ice (or O/N if the protein concentration is too low).
3. Spin tube in microcentrifuge at 14K rpm for 15 min.
4. Remove supernatant (above the cloudy layer at the bottom), leaving protein pellet intact. Pellet should be formed from whitish, fluffy ppt.
5. Wash pellet with 200μl cold acetone by flicking gently.
6. Once this has dissolved completely, leave the samples on ice for 5 minutes.
7. Spin tune in microfuge at 14K rpm, 10min.
8. Repeat steps 4-6 for a total of 2 acetone washes.
9. Carefully remove the supernatant.
8. Dry pellet by placing tube at room temperature for about 10 minutes.
9. For SDS-PAGE, add 1X Invitrogen LDS sample buffer (w/ 100mM final conc. of DTT) and boil sample for 10 min before loading sample onto polyacrylamide gel.
I am doing IP (w/ a-flag Ab) and then elution with 3x flag peptide. Then to concentrate the IP'd proteins, I am trying TCA precipitation. But someone said that it is really hard to dissolve pelleted protein with 1x sample buffer for SDS-PAGE. Right now I am at step 2 (O/N).
Below is my protocol and does anyone have good idea to dissolve pellet (at step 8)?
Many thanks to all of you!
1. Add 1 volume of TCA stock to 4 volumes of protein sample.
2. Incubate 15 min on ice (or O/N if the protein concentration is too low).
3. Spin tube in microcentrifuge at 14K rpm for 15 min.
4. Remove supernatant (above the cloudy layer at the bottom), leaving protein pellet intact. Pellet should be formed from whitish, fluffy ppt.
5. Wash pellet with 200μl cold acetone by flicking gently.
6. Once this has dissolved completely, leave the samples on ice for 5 minutes.
7. Spin tune in microfuge at 14K rpm, 10min.
8. Repeat steps 4-6 for a total of 2 acetone washes.
9. Carefully remove the supernatant.
8. Dry pellet by placing tube at room temperature for about 10 minutes.
9. For SDS-PAGE, add 1X Invitrogen LDS sample buffer (w/ 100mM final conc. of DTT) and boil sample for 10 min before loading sample onto polyacrylamide gel.
When you add the sample buffer, sometimes you see that the buffer could change colour from blue to yellow (due to pH change to acidic), that is caused by TCA. I normally add drops of Tris 1M pH 8 in until it restores the blue colour. Pipeting up and down or vortex before boiling could also help dissolving the protein. But mostly the pellet is hard to dissolve because TCA is not washed away well enough. I think that you will see after first precipitatiion, the pellet is bigger than after washing. Acetone washes help to remove TCA and make dissolving the pellet easier. The boiling part would also help to dissolve more proteins. Also, if it is not dissolve totally, try to mix well and load. It should be OK
try alternative, non-acidic precipitation methods such with acetone or methanol/chloroform