Smolke:Protocols/Heme measurements: Difference between revisions
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#* If you put the replicate tube at 4 °C, the oxalic acid will crash out of solution again. 1 M oxalic acid is soluble at RT but not 4 °C. | #* If you put the replicate tube at 4 °C, the oxalic acid will crash out of solution again. 1 M oxalic acid is soluble at RT but not 4 °C. | ||
#Spin all tubes for 2 minutes at 16000 g. | #Spin all tubes for 2 minutes at 16000 g. | ||
#Transfer 200 μL of each sample to a black 96-well plate and measure the fluorescence. | #Transfer 200 μL of each sample to a black 96-well plate and measure the fluorescence (excite at 400 nm and measure emission at 620 nm). | ||
*For a standard curve: Prepare extra cell samples as above. Add hemin (diluted into water to the desired concentration) to samples, either before or after lysis (measurements are similar). 100 μL/g is approximately the upper end of the linear range. | *For a standard curve: Prepare extra cell samples as above. Add hemin (diluted into water to the desired concentration) to samples, either before or after lysis (measurements are similar). 100 μL/g is approximately the upper end of the linear range. |
Latest revision as of 03:51, 11 October 2011
OverviewThis protocol is used to measure intracellular heme concentrations. Note that it measures total heme levels (not free heme - most if not all protein-bound heme is liberated during the assay). The protocol is derived from the assay described by Sassa (reference below). ProcedureMaterials
Method
Analysis
References
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