McClean:Phluorin Calibration

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==Overview==
==Overview==
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This protocol is for calibrating the ratio of  
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Ratiometric pHluorin is a pH-sensitive GFP derivative.  See Miesenbock, ''et al'' 1998 for the original description of this protein.  Wild-type green fluorescent protein (GFP) exists in two conformations and therefore has a bimodal excitation spectrum with peaks at 395nm and 475nm.  The authors took advantage of these conformation states, and with some clever amino-acid substitutions facilitated pH-dependent switching between the states.  Thus, the relative emission of pHluorin when excited at 395nm vs 475nm is dependent on pH.
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This protocol describes how to measure a calibration curve for pHluorin in your yeast strain of interest.  The relative emission at 395nm or 475nm excitation is measured for permeabilized cells in buffers over a range of pH.  This protocol was adapted from Orij 2009.  See their paper (Figure 2a) for a good example of what your calibration curve should look like.
==Materials==  
==Materials==  
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* Strains of interest to check for mating type
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* Strain of interest
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* yMM421 (aka DBY7730, RC634a) MATa ade2 his6 met1 ura1 can1 cyh1 rme sst1-3
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* Low Fluorescence Media () with appropriate amino acids for your strain
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* yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2
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* Digitonin (Acros Organics, #407565000)
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* YPD plates
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* Phosphate Buffered Saline, pH 7
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* Buffers (see below)
==Protocol==
==Protocol==
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* Let streaks grow 2 days 30°C.
 
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* Inoculate overnight YPD cultures of the mating type testers.
 
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* Dilute the overnights 1:10 with sterile media or water.
 
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* Spread ~200 μl lawn on YPD plates, one for each mating type.
 
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* Incubate 30°C for 30 minutes. 
 
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* Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh toothpick for each plate.  Do NOT pin onto tester plates thate are still wet.  This will just cause your patches to run all over the plate.
 
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* Incubate 30°C overnight.
 
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* Score whether or not the patch has a halo of space around it. If it does, that means that the lawn strain responded to the pheromone emitted by the patch, and thus that they are of opposite mating type. So, if a halo formed around the patched strain on the a tester plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester plates is that you can double-check your scoring by making sure there is a halo on only one tester plate.
 
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==References==
 
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Adapted from Maitreya Dunham's protocol ([http://dunham.gs.washington.edu/protocols.shtml| Dunham Lab Protocols]) which was adapted from Katja Schwartz's protocol from the Botstein lab ([http://www.princeton.edu/genomics/botstein/protocols/| Botstein Lab Protocols])
 
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The strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for
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documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations
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==References==
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make them super-sensitive to pheromone. When exposed to pheromone of the opposite
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* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195
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mating type, the cells arrest.
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* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278
==Notes==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!  Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!  Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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*[[User:Megan N McClean|Megan N McClean]] 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strainStrains of the opposite mating type have not been forming scorable halos.  We may want to try fresh stock from the Botstein lab's -80°C stock.  Alternatively, it seems to work 'sometimes' and since none of us are that careful about how many cells we seed or what growth phase they are in when we do so, perhaps we should play around with that and then be consistent in the future.
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*[[User:Megan N McClean|Megan N McClean]] 10:33, 5 June 2012 (EDT)Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter)Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.
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==References==
 
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* Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195
 
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* Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278
 
==Contact==
==Contact==
<!--Change the information below to your info if you add a new protocol-->
<!--Change the information below to your info if you add a new protocol-->
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 03 November 11(EDT)'''
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*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 05 June 12(EDT)'''
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Revision as of 09:33, 5 June 2012


Contents

Overview

Ratiometric pHluorin is a pH-sensitive GFP derivative. See Miesenbock, et al 1998 for the original description of this protein. Wild-type green fluorescent protein (GFP) exists in two conformations and therefore has a bimodal excitation spectrum with peaks at 395nm and 475nm. The authors took advantage of these conformation states, and with some clever amino-acid substitutions facilitated pH-dependent switching between the states. Thus, the relative emission of pHluorin when excited at 395nm vs 475nm is dependent on pH.

This protocol describes how to measure a calibration curve for pHluorin in your yeast strain of interest. The relative emission at 395nm or 475nm excitation is measured for permeabilized cells in buffers over a range of pH. This protocol was adapted from Orij 2009. See their paper (Figure 2a) for a good example of what your calibration curve should look like.

Materials

  • Strain of interest
  • Low Fluorescence Media () with appropriate amino acids for your strain
  • Digitonin (Acros Organics, #407565000)
  • Phosphate Buffered Saline, pH 7
  • Buffers (see below)


Protocol

References

  • Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins Nature 394 192-195
  • Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccaromyces cerevisiae reveals a relation between intracellular pH and growth Microbiology 155 268-278


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Megan N McClean 10:33, 5 June 2012 (EDT)Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.


Contact

or instead, discuss this protocol.

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