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No binding heat in Substrate and Enzyme ITC

itc substrate enzyme titration

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#1 biobunny

biobunny

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Posted 12 March 2012 - 07:21 PM

Dear all,

I did the ITC (isothermal titration calorimetry) between an enzyme with its known substrate on Microcal ITC200. The concentartion of the ligand (substarte) is 1mM; and the concentration of the enzyme is 100 uM; 1.8 ul/injection X 22 injection;

However, there is no heat of binding observed in the raw data (isotherms). (dilution of substrate(substrate to buffer control) seems to consume some heat; dilution of protein (buffer to protein) seems to produce some heat); while titrating substrate to enzyme seems to be the sum of heats from the two controls.

About the buffer: both the substrate and the enzyme are in 10mM HEPES, 300 uM ZnCl2 and 4% DMSO;

I did not expect that I cannot even observe the binding curve in titration between the enzyme and its substrate!!

Anyone could help???Posted Image Posted Image Posted Image


P.S. the data is attached

Attached Thumbnails

  • itc-question.png






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