BCA assay for reducing ends - (Aug/17/2009 )
this section seems to be one of the less visited sub-sections on this forum, so I am not sure how many people read it on a regular basis. but, I will try asking my question anyway.
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?
BCA is a protein assay, not for sugars!