Team:Washington/Protocols/Cell Lysate Assay

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=<big> Cell Lysate Assay </big>=
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1. Prepare stocks for protease inhibitor, DNAse, and lysozyme.
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*a. Protease Inhibitor = 25x stock ; one tablet into 2ml of buffer
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=Whole Cell Lysate Assay=
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*b. DNAses = 100x stock (10mg/ml initial → 0.1mg/mL final)
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*c. Lysozyme = 10x stock (10mg/ml initial → 1mg/mL final)
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<html>
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2. Resuspend 50ml of each type of cell culture in 1ml of this mixture: 850ul sodium phosphate buffer, 40ul protease inhibitor, 10ul DNAse (0.1mg/ml final), and 100ul lysozyme (1mg/ml final).If testing multiple reactions, just multiply the volumes and divide into eppendorf tubes so that each reaction gets 1mL of cell suspension.
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<script type="text/javascript">
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*a. Buffer: 1ml = 1000ul - (40+10+100) → 850ul 
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$(function() {
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*b. Protease inhibitor: 25x (xml) = 1x (1ml) → 40ul
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  var mainimg = document.getElementById("mainimg");
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*c. DNAses: (10mg/ml)Xul = (0.1mg/ml)1ml  → 10ul
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  var default_src = mainimg.src;
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*d. Lysozyme: (1mg/ml)Xul = (1mg/ml) 1ml → 100ul
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  var restore_mainimg = function() {
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3. At the cell-lysis part, add 111uL 10% triton (1% final) and mix the tube at room temperature for 30 min. Then, centrifuge the tubes at max. speed for 15 min (or until the liquid is very clear and not cloudy.
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    mainimg.src = default_src;
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*x/(1000 +x) = 1/10 ….................. x = 111ul
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  };
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4. Transfer the liquid on top to new labeled eppendorf tube (leave the pellet of lysed stuff; our protein should be sufficiently soluble in aqueous solutions), tube perhaps containing master mix to initiate reaction.
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  $(document.getElementById("area_target")).hover(function() {
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5. Place Target polyspring inserts into the Target DP glass vials.
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    mainimg.src = "/wiki/images/1/1d/Main_graphic2_target.png";
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6. Cell Lysis
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  }, restore_mainimg);
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*a. Sonicate --use micro tip, 60%, 30 on 30 off (POST-assay: cell lysate not so effective)
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  $(document.getElementById("area_secretion")).hover(function() {
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*b. Bugbuster --Use 10x bugbuster, follow protocol (POST-assay: cell lysate not so effective)
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    mainimg.src = "/wiki/images/2/23/Main_graphic2_secretion.png";
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*c. Triton X-100 --Use 1% triton (POST-assay: most effective for cell lysate)
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  }, restore_mainimg);
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  $(document.getElementById("area_display")).hover(function() {
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    mainimg.src = "/wiki/images/a/aa/Main_graphic2_display.png";
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  }, restore_mainimg);
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  $(document.getElementById("area_release")).hover(function() {
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    mainimg.src = "/wiki/images/e/ed/Main_graphic2_release.png";
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  }, restore_mainimg);
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});
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</script>
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</html>
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
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Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.
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*'''Triton Lysis'''
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**Triton Lysis Buffer (10mL, enough for 2 plates)
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***9mL of 1x PBS
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***10mg of Lsyozyme (5-20 is fine)
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***5mg of DNase (2-10 is fine)
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***1mL of 10% Triton X100
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**Add 50uL of Triton lysis buffer to each well
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**Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes
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**Add 250uL of 100mM NaOAc pH 4.0 to each well
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**Spin 40 minutes 4000rpm
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*'''Sonication Lysis'''
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**Add 300uL of 100mM NHAc pH 4.0 to each well
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***No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA
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**Use Plate Sonicator (Ask Chris)
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**Spin 40 minutes 4000rpm
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*'''2.Assay'''
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**Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
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**Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)
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***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
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**Monitor the reaction with the SpectraMax
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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<div style="text-align:center">
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'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''
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&nbsp; &nbsp; &nbsp;
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</div>

Latest revision as of 01:04, 23 September 2011


Whole Cell Lysate Assay

Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.

  • Triton Lysis
    • Triton Lysis Buffer (10mL, enough for 2 plates)
      • 9mL of 1x PBS
      • 10mg of Lsyozyme (5-20 is fine)
      • 5mg of DNase (2-10 is fine)
      • 1mL of 10% Triton X100
    • Add 50uL of Triton lysis buffer to each well
    • Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes
    • Add 250uL of 100mM NaOAc pH 4.0 to each well
    • Spin 40 minutes 4000rpm


  • Sonication Lysis
    • Add 300uL of 100mM NHAc pH 4.0 to each well
      • No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA
    • Use Plate Sonicator (Ask Chris)
    • Spin 40 minutes 4000rpm


  • 2.Assay
    • Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
    • Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)
      • Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
    • Monitor the reaction with the SpectraMax



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