Smolke:Protocols/Heme measurements
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(New page: {{Smolke_Top}} ==Overview== This 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...) |
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===Method=== | ===Method=== | ||
#Grow cells to desired density. Ideally, you want roughly 8 OD*mL of yeast (e.g. 20 mL at OD 0.4). Measure the density of each sample. | #Grow cells to desired density. Ideally, you want roughly 8 OD*mL of yeast (e.g. 20 mL at OD 0.4). Measure the density of each sample. | ||
| - | #Pellet cells (in a Falcon tube), 5 minutes at 4 °C and 5000 g. Wash with water, transfer to an amber Eppendorf tube, and repellet 5 minutes at 4 °C and 8000 g. | + | #Pellet cells (in a Falcon tube), 5 minutes at 4 °C and 5000 g. Wash with water, transfer to an amber Eppendorf tube, and repellet for 5 minutes at 4 °C and 8000 g. |
#Discard supernatant, resuspend in 500 μL of 20 mM oxalic acid. Store in a closed box in the fridge overnight (16-24 hours). | #Discard supernatant, resuspend in 500 μL of 20 mM oxalic acid. Store in a closed box in the fridge overnight (16-24 hours). | ||
#Add an equal volume (500 μL) of 2 M oxalic acid. Pipet to mix, then transfer one volume (500 μL) to a new Eppendorf tube. | #Add an equal volume (500 μL) of 2 M oxalic acid. Pipet to mix, then transfer one volume (500 μL) to a new Eppendorf tube. | ||
#* 2 M oxalic acid is not soluble in water at room temperature. You must heat the oxalic acid to dissolve it before starting the protocol | #* 2 M oxalic acid is not soluble in water at room temperature. You must heat the oxalic acid to dissolve it before starting the protocol | ||
#Transfer the first amber Eppendorf tube to a heat block at 95-98 °C. Heat for 30 minutes. Meanwhile, store the replicate tube in a closed box at RT. | #Transfer the first amber Eppendorf tube to a heat block at 95-98 °C. Heat for 30 minutes. Meanwhile, store the replicate tube in a closed box at RT. | ||
| - | #* If you put the replicate tube at 4 °C, the oxalic acid will crash out of solution again. 1 M 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. | ||
| - | + | *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). | + | |
===Analysis=== | ===Analysis=== | ||
| - | + | *For each boiled sample, subtract the fluorescence of the corresponding unboiled sample to correct for background fluorescence. | |
| - | *CSY3 is ~0.53 mg DCW/(OD*mL) | + | *Remember to correct for the effect of variable sample concentrations. |
| - | *Hans et al. quote a value of 2.38 mL/g DCW as the intracellular volume of ''S. cerevisiae'' (Han et al., 2001). | + | *To estimate intracellular concentrations: |
| - | **We arrive at similar numbers (within a factor of 1.5) by reasoning from first principles (cell number per OD and average cell size). | + | **CSY3 is ~0.53 mg DCW/(OD*mL) |
| - | *For 8 OD*mL, this gives an approximate intracellular volume of 10μL. | + | **Hans et al. quote a value of 2.38 mL/g DCW as the intracellular volume of ''S. cerevisiae'' (Han et al., 2001). |
| + | ***We arrive at similar numbers (within a factor of 1.5) by reasoning from first principles (cell number per OD and average cell size). | ||
| + | **For 8 OD*mL, this gives an approximate intracellular volume of 10μL. | ||
*Reproducibility between extracts of a given sample is ~10%, similar to the reproducibility between samples. | *Reproducibility between extracts of a given sample is ~10%, similar to the reproducibility between samples. | ||
==References== | ==References== | ||
| - | Hans MA, Heinzle E, & Wittmann C (2001) Quantification of intracellular amino acids in batch cultures of ''Saccharomyces cerevisiae''. Applied microbiology and biotechnology 56(5):776-779. | + | #Hans MA, Heinzle E, & Wittmann C (2001) Quantification of intracellular amino acids in batch cultures of ''Saccharomyces cerevisiae''. Applied microbiology and biotechnology 56(5):776-779. |
| - | Sassa S (1976) Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells. The Journal of experimental medicine 143(2):305-315. | + | #Sassa S (1976) Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells. The Journal of experimental medicine 143(2):305-315. |
==Contact== | ==Contact== | ||
[[Josh Michener]] | [[Josh Michener]] | ||
Revision as of 06:06, 11 October 2011
