I am trying to standardize luciferase assay so I am using 1.6uM DnaK, 320nM dnaJ, and 800nM GrpE, to refold 10uM of thermally[30 degree for 10 min] denatured luciferase[final conc 80nM in the refolding mixture] in the final refolding soln volume of 125uL. Right after adding denatured luc in the refolding solution, I am taking the luminescence at different timepoints by adding 1ul of the soln to 40 uL of luciferin [promega]. I am adding luciferin to the multiplate reader plate amd manually adding 1ul of the refolding mix to it and taking the reading. The problems I am facing are
1) the intensity i am observing is very low [100-500 RLU] as compared to stated in papers. [Synergy H1- plate reader is used]
2) the graph I am observing is not following the sigmoidal trend and is having a very high standard deviation.
where am I going wrong?
I would be concerned about the reproducibility of adding 1 µl of the fairly concentrated protein solution. Larger volumes can be added with better reproducibility. Also, make sure the solutions are well-mixed in the plate by using a plate mixer after making additions to the wells.
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