Freimoser:Protocols:PolyP1: Difference between revisions

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General comment:<br /> Poly P can be purified on Qiagen PCR-product purification columns (either individual spin columns or for example 96-well plates). The columns can be reused after recycling according to the protocol given below.
General comment:<br /> Poly P can be purified on Qiagen PCR-product purification columns (either individual spin columns or for example 96-well plates). The columns can be reused after recycling according to the protocol given below. The Reference for the method is:<br>
Werner TP, Amrhein N, Freimoser FM (2005). Novel method for the quantification of inorganic polyphosphate (iPoP) in ''Saccharomyces cerevisiae'' shows dependence of iPoP content on the growth phase. ''Archives of Microbiology'' (2005) 184: 129-136 (DOI 10.1007/s00203-005-0031-2). [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16184370&query_hl=1&itool=pubmed_docsum PubMed]


==Extraction and purification of poly P from yeast==
==Extraction and purification of poly P from yeast==


# Pellet 0.5 or 1 OD<sub>600</sub> Unit of yeast cells in a 1.5 ml Eppi. (12000 rpm, 1 min), discard the supernatant.
# Pellet 0.5 or 1 OD<sub>600</sub> unit of yeast cells in a 1.5 ml tube (13'000 rpm, 1 min), discard the supernatant.
# Add exactly 50 μl of 1 M sulfuric acid and vortex vigorously for 15 seconds (This extract can be stored at -20°C if necessary).
# Add exactly 50 μl of 1 M H<sub>2</sub>SO<sub>4</sub> and vortex vigorously for 15 seconds (this extract can be stored at -20°C if necessary).
# Add exactly 50 μl of 2 M NaOH to the extract to neutralize the pH.
# Add exactly 50 μl of 2 M NaOH to neutralize the extract.
# Add 100 μl of 1M Tris-Malate buffer pH 7.5 with 6% Neutral red solution as an indicator for the pH. The solution should be red or dark orange. (neither pink nor yellow)
# Add 100 μl of 1M Tris-Malate buffer (pH 7.5, with 6% Neutral red solution as a pH indicator). The solution should be red or dark orange (neither pink nor yellow).
# Centrifuge (13000 rpm, 1 min) and transfer the supernatant to a new tube.
# Centrifuge (13'000 rpm, 1 min) and transfer the supernatant to a new tube.
# Add 600 ul of 6 M NaI and mix.
# Add 600 μl of 6 M NaI and mix.
# Fill the mixture in the purification plate and apply vacuum for 1 min.
# Transfer the mixture to the purification plate (or the spin column) and apply vacuum (or spin) for 1 min.
# Wash with 750 ul of washing buffer and apply vacuum for 1 min.
# Wash with 750 μl of washing buffer and apply vacuum (or spin) for 1 min.
# Repeat this step, but apply vacuum for 10 min.
# Repeat this step, but apply vacuum for 10 min (or spin twice).
# Remove residual washing buffer on outside of plate.
# Remove residual washing buffer on outside of plate.
# Add 100 ul of ddH2O (which must not contain any phosphate!) and let the plate stand for one minute before elution by vacuum for 2 min.
# Add 100 μl of H<sub>2</sub>O (which must not contain any phosphate!) and let the plate/columns stand for one minute before elution by vacuum (or centrifugation) for 2 min.
# After use wash columns with 1 ml ddH2O.<br />
# After use wash columns with 1 ml H<sub>2</sub>O.<br />


==Quantification of poly P==
==Quantification of poly P==


# Prepare a master mix including 10 ul of 10X buffer, 1 ul (0.2 ug) of Polyphosphatase from S.cerevisiae and 39 ul of phosphate free ddH2O for each sample.
# Prepare a master mix including 10 μl of 10X buffer, 0.1 l (0.02 μg) of polyphosphatase from S.cerevisiae and 39.9 ul of phosphate free ddH2O for each sample.
# Fill 50 ul of this master mix to a 96 well flat bottom microtiter plate and add 50 ul from your eluted iPoP samples.
# Fill 50 μl of this master mix to a 96 well flat bottom microtiter plate and add 50 μl from your eluted iPoP samples.
# Incubate for 1-2 hours at 37°C.
# Incubate for 1-2 hours at 37°C.
# Prepare a standard curve for Pi detection: Add 0, 5, 10, 20, 40, 60, 80 and 100 ul of 100 uM NaH2PO4 (Pi) to your microtiter plate and fill up to 100 ul with ddH2O.
# Prepare a standard curve for Pi detection: Add 0, 5, 10, 20, 40, 60, 80 and 100 μl of 100 μM NaH<sub>2</sub>PO<sub>4</sub> (Pi) to your microtiter plate and fill up to 100 μl with H<sub>2</sub>O.
# Prepare a 1:3 dilution of your samples: Add 50 ul of ddH2O to your samples, mix by pipetting and transfer 50 ul to a new microtiter plate containing 100 ul ddH2O per well. Mix again by pipetting and discard 50 ul solution from the diluted samples. Do the same to your Pi standart curve.
# Prepare a 1:3 dilution of your samples: Add 50 μl of H<sub>2</sub>O to your samples, mix by pipetting and transfer 50 μl to a new microtiter plate containing 100 μl H<sub>2</sub>O per well. Mix again by pipetting and discard 50 μl solution from the diluted samples. Do the same to your Pi standart curve. This dilution is only necessary if the polyphosphate content of your samples is high.
# Mix 86 ul of Molybdate solution and 64 ul of Malachite green solution per sample and add 150 ul to each sample.
# Mix 86 μl of Molybdate solution and 64 μl of Malachite green solution per sample and add 150 μl to each sample.
# Use a 96 plate reader to determine the OD at 595 nm. (590 – 650 is a possible range)
# Use a 96 plate reader to determine the OD at 595 nm. (590 – 650 is a possible range)


==Recycling of Qiagen-columns for poly P purification==
==Recycling of Qiagen-columns for poly P purification==


# Add 500 ul of 0.2 M Acetic acid (0.6ml in 49.4ml ddH2O) and apply vacuum for 1 min.
# Add 500 μl of 0.2 M Acetic acid (0.6ml in 49.4ml H<sub>2</sub>O) and apply vacuum for 1 min.
# Add 500 ul of 0.05 M EDTA pH 8 and apply vacuum for 1 min.
# Add 500 μl of 0.05 M EDTA (pH 8) and apply vacuum for 1 min.
# Add 1 ml of ddH2O and apply vacuum for 1 min.
# Wash twice with 1 ml H<sub>2</sub>O and apply vacuum for 1 min.
# Repeat last step.


{| width="100%" cellpadding="5"
==Solutions==
|
 
[http://creativecommons.org/licenses/by-nc-nd/2.0/ [[Image:somerights20.gif|Creative Commons License]]]<br />
====Binding buffer====
| class="small" |
*6 M NaI (Natriumiodid)
<div style="text-align: justify"><font size="-2">All content on this site, including all images and drawings, are licensed under a [http://creativecommons.org/licenses/by-nc-nd/2.0/ Creative Commons License] except when noted. Commercial use or alteration of any material from this site is illegal without a written permission from Florian Freimoser. Any content from this site may be distributed for non-commercial purposes provided the authorship is clearly acknowledged.</font></div>
 
|}
====Wash buffer====
*50% Ethanol
*100 mM NaCl
*10 mM Tris (pH 7)
*1 mM EDTA
====Neutral red solution====
*0.05 g/50 ml neutral red
*70% Ethanol
 
====10X Buffer for Polyphosphatase====
*50 mM M Tris (pH 7)
* 5 mM MgCl<sub>2</sub>
 
====Malachite green solution====
*0.27 g malachite green
*1.4 g polyvinylalcohol (100'000, Fluka: 81386)
*fill up to 400 ml with H<sub>2</sub>O
*Polyvinylalcohol dissolves very slowly. Usually we stirr the solution overnight.
 
====Molybdate solution====
*1.384 g ammonium molybdate
*4.48 ml concentrated H<sub>2</sub>SO<sub>4</sub>
*fill up to 40 ml with H<sub>2</sub>O

Latest revision as of 05:16, 27 June 2007

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General comment:
Poly P can be purified on Qiagen PCR-product purification columns (either individual spin columns or for example 96-well plates). The columns can be reused after recycling according to the protocol given below. The Reference for the method is:
Werner TP, Amrhein N, Freimoser FM (2005). Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase. Archives of Microbiology (2005) 184: 129-136 (DOI 10.1007/s00203-005-0031-2). PubMed

Extraction and purification of poly P from yeast

  1. Pellet 0.5 or 1 OD600 unit of yeast cells in a 1.5 ml tube (13'000 rpm, 1 min), discard the supernatant.
  2. Add exactly 50 μl of 1 M H2SO4 and vortex vigorously for 15 seconds (this extract can be stored at -20°C if necessary).
  3. Add exactly 50 μl of 2 M NaOH to neutralize the extract.
  4. Add 100 μl of 1M Tris-Malate buffer (pH 7.5, with 6% Neutral red solution as a pH indicator). The solution should be red or dark orange (neither pink nor yellow).
  5. Centrifuge (13'000 rpm, 1 min) and transfer the supernatant to a new tube.
  6. Add 600 μl of 6 M NaI and mix.
  7. Transfer the mixture to the purification plate (or the spin column) and apply vacuum (or spin) for 1 min.
  8. Wash with 750 μl of washing buffer and apply vacuum (or spin) for 1 min.
  9. Repeat this step, but apply vacuum for 10 min (or spin twice).
  10. Remove residual washing buffer on outside of plate.
  11. Add 100 μl of H2O (which must not contain any phosphate!) and let the plate/columns stand for one minute before elution by vacuum (or centrifugation) for 2 min.
  12. After use wash columns with 1 ml H2O.

Quantification of poly P

  1. Prepare a master mix including 10 μl of 10X buffer, 0.1 l (0.02 μg) of polyphosphatase from S.cerevisiae and 39.9 ul of phosphate free ddH2O for each sample.
  2. Fill 50 μl of this master mix to a 96 well flat bottom microtiter plate and add 50 μl from your eluted iPoP samples.
  3. Incubate for 1-2 hours at 37°C.
  4. Prepare a standard curve for Pi detection: Add 0, 5, 10, 20, 40, 60, 80 and 100 μl of 100 μM NaH2PO4 (Pi) to your microtiter plate and fill up to 100 μl with H2O.
  5. Prepare a 1:3 dilution of your samples: Add 50 μl of H2O to your samples, mix by pipetting and transfer 50 μl to a new microtiter plate containing 100 μl H2O per well. Mix again by pipetting and discard 50 μl solution from the diluted samples. Do the same to your Pi standart curve. This dilution is only necessary if the polyphosphate content of your samples is high.
  6. Mix 86 μl of Molybdate solution and 64 μl of Malachite green solution per sample and add 150 μl to each sample.
  7. Use a 96 plate reader to determine the OD at 595 nm. (590 – 650 is a possible range)

Recycling of Qiagen-columns for poly P purification

  1. Add 500 μl of 0.2 M Acetic acid (0.6ml in 49.4ml H2O) and apply vacuum for 1 min.
  2. Add 500 μl of 0.05 M EDTA (pH 8) and apply vacuum for 1 min.
  3. Wash twice with 1 ml H2O and apply vacuum for 1 min.

Solutions

Binding buffer

  • 6 M NaI (Natriumiodid)

Wash buffer

  • 50% Ethanol
  • 100 mM NaCl
  • 10 mM Tris (pH 7)
  • 1 mM EDTA

Neutral red solution

  • 0.05 g/50 ml neutral red
  • 70% Ethanol

10X Buffer for Polyphosphatase

  • 50 mM M Tris (pH 7)
  • 5 mM MgCl2

Malachite green solution

  • 0.27 g malachite green
  • 1.4 g polyvinylalcohol (100'000, Fluka: 81386)
  • fill up to 400 ml with H2O
  • Polyvinylalcohol dissolves very slowly. Usually we stirr the solution overnight.

Molybdate solution

  • 1.384 g ammonium molybdate
  • 4.48 ml concentrated H2SO4
  • fill up to 40 ml with H2O
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