Problem with TCA precipitation. - can't see pellet, what should i do..VERY URGENT..!!!!!&# (Jan/21/2008 )
Hi everyone,
i am following TCA precipitation protocol forsample preparation for 2-Dimensional electrophoresis.
1. Defrost the sample ovcer ice.
2. Add equal volume of 20% w/v TCA inacetone containing 0.07% beta-mercaptoethnolfor at leat 1 hr at 4degree C.
3. Then pellet protein ( 13000 rpm for 10 min) at 4degree then aspirate supernatant.
4. Wash pellet at least twice with cold acetone (keep acetone at -20 degree) containing 0.07% beta- ME to remove residual TCA.
5 Remove the residual action by air drying (~ 1 min) and resuspend pellet in buffer of choice.
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as i am following the same protocol but could not get any pellet after centrifugation. what do i do now.
and if any one has some another protocol then kindly let me know.
i will be very much thankfull to you for this act of kindness.
waiting for reply
i am following TCA precipitation protocol forsample preparation for 2-Dimensional electrophoresis.
1. Defrost the sample ovcer ice.
2. Add equal volume of 20% w/v TCA inacetone containing 0.07% beta-mercaptoethnolfor at leat 1 hr at 4degree C.
3. Then pellet protein ( 13000 rpm for 10 min) at 4degree then aspirate supernatant.
4. Wash pellet at least twice with cold acetone (keep acetone at -20 degree) containing 0.07% beta- ME to remove residual TCA.
5 Remove the residual action by air drying (~ 1 min) and resuspend pellet in buffer of choice.
-----------------------------------------------------------------------------------------------------------------
as i am following the same protocol but could not get any pellet after centrifugation. what do i do now.
and if any one has some another protocol then kindly let me know.
i will be very much thankfull to you for this act of kindness.
waiting for reply
at which step the pellet disappears?
was the pellet invisible? try to resuspend the putative pellet and check the protein concentration...
The answer to the previous question would be useful, but for TCA precipitation I add 100% TCA in water to my sample to reach a final concentration of 10% TCA. THen I incubate on ice for 30 minutes and spin the sample at 4 deg C at 10,000g for 15 minutes. I then finish up with acetone washes and then drying as you suggest. Lastly, I resuspend in a alkaline buffer (pH 8) to neutralize the remaining TCA.
IF you don't see at least some cloudiness in your sample after adding the TCA, you might just have low protein quantity. I hope this helps.