ADP/ATP Ratio Assay in M. tuberculosis

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We tested ab65313 ADP/ATP Ratio Assay in M. tuberculosis (Mtb) strains H37Rv and 18b. The main goal was to determine if the Nucelotide Releasing Buffer (NRB) was able to cause release of nucleotides from Mtb with or without additional disruption (bead beating).
Experimental set-up:
Cells: Mtb strains H37Rv and 18b were grown in 7H9 media and harvested at an OD600 of ~0.4. 175 Million total bacteria were used from each strain.
Control: 7H9 alone
Treatments:
- "Lysis": NRB (5-10 minutes)
- "Beads": NRB (5-10 minutes) + bead-beading (speed 6.60 - 3 x 30s runs)
Measurement:
ab65313 on a Pherastar Plus plate reader on bioluminescent setting
Standard Curve:
ATP disodium salt (ab120385) dilutions to create a standard curve.
Results:
Standard curve: log10(RLU) = 3.739 + 0.938 * log10(ATPconc)
Boxplot of ADP and ATP concentrations on two technical replicates on each of two biological replicates. #N.B. The gain setting on the luminometer resulted in ADP readings (Data D of the kit) for the bead-beating groups that were off-scale and thus non-quantifiable, but still plotted in the attached figure.
Discussion:
NRB alone did not lead to appreciable release of nucleotides from two strains of Mtb. The addition of bead beating to NRB led to significant increases in measured concentrations of both nucleotides in both strains.

Mr. Gregory Olson

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Submitted Oct 06 2017

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