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TCA/acetone precipitation - no visible pellet for 500 micrograms !

TCA precipitation

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#1 zakarino

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Posted 18 January 2012 - 01:53 AM

Good morning all,

I am trying to develop some protein precipitation on BSA solution before using precious samples.

I am using 200 uL of 2.5 ug/uL of BSA in distilled water (solution not buffered). I have tried several things:

- add 400 uL of cold (-20C) TCA 10% in Acetone for 45 min ( so that's final concentration of 6.6%) at -20C
- centrifuge for 15 min at 21000g at 4C

OR

- add 1mL of cold (-20C) TCA 12% or 18% in Acetone overnight at -20C (so that's final concentrations of 10 and 15%)
- centrifuge for 15 min at 21000g at 4C

And I don't have any pellet at this time, I then wash twice with cold acetone and when I do a protein assay or a coomassie blue staining I don't have anything. Thus I believe I don't precipitate as I don't see a pellet or else the pellet is not visible and I discard it when I discard supernatant after centrifugation steps.

The step seems easy so I must do something wrong. Anyone could suggest something or identify where I could possibly go wrong?

Cheers,

Zakarino

#2 knuf

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Posted 18 January 2012 - 06:08 AM

When I do TCA precipitations I add 100% TCA so that the final concentration is 20%. I let that stir end-over-end in the cold room for 10 minutes then centrifuge at max speed in our microfuge for 10 minutes.

#3 zakarino

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Posted 19 January 2012 - 04:58 AM

Hi,

Thank you for your advice.

I noticed something which raises more questions.

I repeated the experiment with 10% TCA final concentration with 5 mg of BSA (as I am sure this content should result in a pellet). When I add TCA in acetone, a white precipitate forms, after wash steps in acetone and resolubilisation and loading in SDS-PAGE, I have a very nice staining and I can demonstrate efficient precipitation and recovery if compared with BSA samples not precipitated.

However, if I vortexed just after adding the TCA 10%, the precipitate disappeared and no pellet was formed and recovered after centrifugation....

Has anyone ever noticed such things?

I will try to scale it down to see if I can detect a pellet with lower amounts of BSA...

Bye

#4 Anupa

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Posted 24 August 2012 - 12:17 PM

I would like to know in what usually you resolubilize your pellet





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