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BCA assay for reducing ends


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#1 Sad Cow

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Posted 17 August 2009 - 02:49 PM

Hello,

[ I am also cross-posting this in the Biochemistry sub-section. Please delete this post if the rules do not allow this. The reason why I am doing this is that the BCA assay is most often used for protein quantification, so the people who visit this sub-section might also be able to answer the question. ]

I am doing a simple specific activity assay on a carbohydrate active enzyme of known activity. The enzyme is a glycoside hydrolase (i.e., cleaves a glucomannan backbone).

the incubations were stopped at the appropriate time points (0, 5, 30, etc. minutes) by freezing at -80 degrees celsius.

but, for some reason when I came back after the weekend to complete the BCA assay (thawing the reaction volumes on ice and then adding fresh BCA reagent), the assay completely failed. i.e., all the resulting product vs. time curves were completely flat. they showed no activity at all.

does anyone know how freezing could have adversely affected anything?

the reaction's products are just shorter polysaccharides. would sugars get damaged from freezing? or am I missing something?

thanks.

Edited by Sad Cow, 17 August 2009 - 02:50 PM.


#2 dxm

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Posted 25 August 2009 - 08:13 PM

Just wondering, are you measuring sugar conc. with BCA? I only know BCA can measure protein concentration, if it is so, I think it is normal you have flat curve as BCA assay cannot measure sugar conc.
Plz correct me if I am wrong.

#3 DRT

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Posted 25 August 2009 - 09:51 PM

I think BCA will pick up any reducing ends, carbohydrate or peptide.
I don’t know the answer to Sad Cow’s OP but a couple of initial thoughts: Did your standard curve work out ok? Could the response from the enzyme be swamping the carbohydrate response?




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