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TCA precipitation - (Jul/21/2005 )

dry.gif I wonder if TCA precipitation can acidify the proteins. I use Clean-Up kit (Amersham). It means 2 step procedure: first- TCA precipitation and the second- acetone wash, and I noticed that from time to time the final pellet after acetone wash and drying can be acidic. I noticed it because I resuspend the pellet in Rehydration buffer for 2D gels. The buffer has bromophenol. Should I wash the TCA pellet with water and then precipitate that suspension with acetone?
If anybody has good experience with TCA precipitation, please, give me a hand.

-Mikki-

I have encountered the same problem when I have TCA ppt proteins using the same protocol.

My bromophenyl blue will turn yellow. What I did was add 2-3ul of 1M tris pH 9 and it neutrilized the acid.

-pBluescript-

hi
in my TCA / acetone protocol, i precipitate and then wash twice with an equal vilume of acetone as the volume of sample at beginning. after pelletting i let dry great and resuspend in 2D rehydratation buffer pipetting down the pellet and let sit 1h RT vortexing every 10'.
2 wash instead of 1 has solved my problem of acidification.

-fred_33-

Wash more with acetone or use 50mM Tris in your re-suspension buffer

-Daniel Tillett-

QUOTE (Mikki @ Jul 21 2005, 07:56 AM)
dry.gif I wonder if TCA precipitation can acidify the proteins. I use Clean-Up kit (Amersham). It means 2 step procedure: first- TCA precipitation and the second- acetone wash, and I noticed that from time to time the final pellet after acetone wash and drying can be acidic. I noticed it because I resuspend the pellet in Rehydration buffer for 2D gels. The buffer has bromophenol. Should I wash the TCA pellet with water and then precipitate that suspension with acetone?
If anybody has good experience with TCA precipitation, please, give me a hand.

-Mikki-

QUOTE (Mikki @ Jul 21 2005, 07:56 AM)
dry.gif I wonder if TCA precipitation can acidify the proteins. I use Clean-Up kit (Amersham). It means 2 step procedure: first- TCA precipitation and the second- acetone wash, and I noticed that from time to time the final pellet after acetone wash and drying can be acidic. I noticed it because I resuspend the pellet in Rehydration buffer for 2D gels. The buffer has bromophenol. Should I wash the TCA pellet with water and then precipitate that suspension with acetone?
If anybody has good experience with TCA precipitation, please, give me a hand.


i agree! i usually wash the pellet AT LEAST 4 times with 500ul acetone.
after drying never had any problems, and bromophenol stays blue as always smile.gif

-Jusu-

i wash the precipitaion pellet twice with acetone and neutralise the resolubilised pellet with some ┬Áls of 1M NaOH till the buffer turns back to blue...

mike

-jadefalcon-