Glycerol 3 phosphate colorimetric assay kit

Klebsiella pneumoniae containing pBAD33, pBAD33::HistaqKPN00353, pBAD33::Histaq353H65Q, pBAD33::Histaq353H65D, pBAD33::Histaq353H65E, pBAD33::Histaq353H65R, or pBAD33::Histaq353H110Q was grown overnight at 37°C in LB medium containing chloramphenicol (100 μg/mL). Then, the pre-cultures were diluted 100-fold in fresh LB medium containing chloramphenicol (100 μg/mL). After the cultures were grown at 37°C in a shaking incubator at 200 rpm for 2 h, 0.4% arabinose was added for protein induction. After induction at 30°C and 150 rpm for 3 h, the bacteria were centrifuged and resuspended in fresh MCM. After 4 h of growth, the cell density was measured by spectrophotometry at 600 nm. An aliquot [OD × V(ml) = 1] of cells was centrifuged and homogenized in 400 μL of G3P assay buffer provided in the Glycerol 3-Phosphate Colorimetric Assay Kit (Sigma–Aldrich, St Louis, MO, United States). The intracellular G3P was then measured according to the manufacturer’s instructions using the Glycerol 3-Phosphate Colorimetric Assay Kit (Sigma–Aldrich, St Louis, MO, United States).

For the analysis of glycerol 3-phosphate (G3P), 10 OD₆₀₀ units of yeast cells were harvested and lysed using the cellLytic Y-Yeast cell Lysis reagent (Sigma–Aldrich). Cells were incubated with 100 μl lysis buffer for 15–30 min at room temperature and centrifuged for 10 min to remove cell debris. For wild type analysis 10–20 μl, and for the analysis of Δgph1 2.5–5 μl of the supernatant were used for the glycerol 3-phosphate assay using the Glycerol 3-phosphate Colorimetric Assay Kit (Sigma-Aldrich).
For qualitative analysis of glycogen, cells were grown to the stationary phase and spotted onto YPD plates. Plates were grown for 48 h at 30°C prior to detection of glycogen by exposing plates to iodine vapor [38 (link)]. The intensity of the brown color was used as indicator for the cellular glycogen content.
Analysis of glycogen was performed following the instructions of the manual of the Glycogen Assay Kit (Sigma–Aldrich). For this purpose, 2 OD₆₀₀ units of yeast cells were harvested and lysed using the cellLytic Y-Yeast cell Lysis reagent (Sigma–Aldrich). Cells were incubated with 20 μl lysis reagent for 15–30 min at room temperature and centrifuged for 10 min to remove cell debris. For the wild type strain 5 μl, and for the Δgph1 strain 2.5 μl of homogenate were used for glycogen analysis.

The concentration of G3P was determined by a commercial Glycerol 3-phosphate Colorimetric Assay Kit (MAK207, Sigma-Aldrich), following the manufacturer’s instructions. P. aeruginosa PAO1 and ΔglpD were grown to an OD_600nm value of 1 in MSM containing 40 mM succinate or glucose as the carbon source. Then, 20 mM glycerol was added, and the strains were cultivated for 1.5 h. Bacterial cultures sampled before and after the glycerol treatment were subjected to G3P quantification. For the extracellular G3P assays, 15 μL of the culture medium supernatant was used for measurement. For the intracellular G3P assays, the cells of P. aeruginosa PAO1 and ΔglpD were centrifuged, washed, and adjusted to an OD_600nm of 7.5 in 200 μL of G3P assay buffer for cell lysis. After centrifugation at 13,000 × g for 5 min, 10 μL of the centrifugal supernatant was used for measurement. The content of G3P was calculated based on the standard curve and was normalized by the sample volume added to the detection system.