Protein Methods Homework Assignments 341)-Compacibility Mode a- 4. You declde to

Question

Protein Methods Homework Assignments 341)-Compacibility Mode a- 4. You declde to measure one of your afflaity column elution fractions for LDH activity Therefore, you add 2 pl of your eluted sample to 998 pl: of your LDH assay mix already in a cuvette, plunge quickly, and record your data. You get a linear line that glves you A of 0.129761 over 3 second time span. What is your activity in jumoles/min and Why do we measure the Ae over time when determining LDH activity? Is it possible that other proteins will bind to the Cibacron Blue affinity columm? why?

Explanation / Answer

Tis can be calculated by beer-lambert law,

[A - absorbance, e- extinction coefficient, c- concentration, l-lenght

A = 0.129761 for 3 min. therefore for 1 min, A=0.04325.

A=e c l, where l= 1cm and e= 6220

therefore, c= A/el

c = 6.9539x10^-6 umoles/min

Since 2ul of eluted sample is added in 998ul if the mix making up to 1ml. therefore,

c = 6.9539x10^-6 x 0.002

=1.39078x10^-8 umoles/min/ml

When determining LDH activity we determine A340 over time, becuase the end product of the reaction is NADH whose extictinction coefficient (e) is taken into consideration , and e for NADH is at A340.

Yes, other proteins will bind to cibacron blue, because the dye might imitate the conformation of the coenzyme and bind to the corresponding binding sites of the respective proteins.

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