Smolke:Protocols/Heme measurements

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Current revision (06:51, 11 October 2011) (view source)
 
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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.

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Contents

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 is liberated during the assay). The protocol is derived from the assay described by Sassa (reference below).

Procedure

Materials

  • 2 M oxalic acid
  • 20 mM oxalic acid
  • Black 96-well plates
  • Hemin standard (~1 g/L in DMSO)

Method

  1. 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.
  2. 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.
  3. Discard supernatant, resuspend in 500 μL of 20 mM oxalic acid. Store in a closed box in the fridge overnight (16-24 hours).
  4. 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
  5. 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 oxalic acid is soluble at RT but not 4 °C.
  6. Spin all tubes for 2 minutes at 16000 g.
  7. 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.

Analysis

  • For each boiled sample, subtract the fluorescence of the corresponding unboiled sample to correct for background fluorescence.
  • Remember to correct for the effect of variable sample concentrations.
  • To estimate intracellular concentrations:
    • CSY3 is ~0.53 mg DCW/(OD*mL)
    • 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.

References

  1. 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.
  2. 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

Josh Michener

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