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Neutralizing 5% TCA


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#1 nueropharm

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Posted 11 May 2011 - 11:12 AM

Hello there,

When I google search "neutralizing TCA" all I get is stuff about baking soda for cosmetic chemical peels or info about tattoo removal.

I'm using neuronal cultures. I need to measure their cAMP production after they were exposed to various treatments. We're going to assay both the NB they were in with treatment, and the 5% TCA they are now in. The cAMP EIA kit from GE Healthcare comes with instructions to neutralize TCA with diethyl ether. My lab manager would prefer a less volatile approach if possible. I thought about 1 M Tris buffer but I wonder if that may interfere with the immunoprecipitation process. Any other ideas?

Thanks.

#2 protolder

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Posted 11 May 2011 - 09:36 PM

Hello there,

When I google search "neutralizing TCA" all I get is stuff about baking soda for cosmetic chemical peels or info about tattoo removal.

I'm using neuronal cultures. I need to measure their cAMP production after they were exposed to various treatments. We're going to assay both the NB they were in with treatment, and the 5% TCA they are now in. The cAMP EIA kit from GE Healthcare comes with instructions to neutralize TCA with diethyl ether. My lab manager would prefer a less volatile approach if possible. I thought about 1 M Tris buffer but I wonder if that may interfere with the immunoprecipitation process. Any other ideas?

Thanks.

Hola,Tris is a biological buffer that is in the storage medium of the majority of pure proteins for activity assay, in order to add less volume possible I woul prepare a Tris base solution wich has a pH of 12 or 13. Other possibility is any alcali as NaOH. . according to the sample volume that you have if itīs very little I would assay the neutralization in big volume in a pHmeter and after reduce the neutarlization agent proportionaly to the relationship betwen both volumes: pHmetr/sample. Buena suerte

#3 nueropharm

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Posted 12 May 2011 - 08:55 AM

[/quote]
Hola,Tris is a biological buffer that is in the storage medium of the majority of pure proteins for activity assay, in order to add less volume possible I woul prepare a Tris base solution wich has a pH of 12 or 13. Other possibility is any alcali as NaOH. . according to the sample volume that you have if itīs very little I would assay the neutralization in big volume in a pHmeter and after reduce the neutarlization agent proportionaly to the relationship betwen both volumes: pHmetr/sample. Buena suerte
[/quote]

Gracias! One question though -- wouldn't all the salts from the Tris interfere with the EIA process?

#4 mdfenko

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Posted 12 May 2011 - 11:47 AM

ethyl ether (aka diethyl ether) is used to extract tca from aqueous solution. using it doesn't actually neutralize the tca, it removes it.

tca is more soluble in ether than in water and ether and water are not miscible. it is also more soluble in alcohol but alcohol is miscible with water so you won't remove it.

if you just neutralize with base then you will have salts remaining which may interfere with later use.

follow the kit's recommendations but work in a explosion-proof fume hood and a room with good ventilation.

Edited by mdfenko, 12 May 2011 - 11:47 AM.

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