Team:Washington/Protocols/Cell Lysate Assay

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==Whole Cell Lysate Assay==  
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=Whole Cell Lysate Assay=
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.
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'''
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***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
**Monitor the reaction with the SpectraMax
**Monitor the reaction with the SpectraMax
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***Basic Settings = ???
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''
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&nbsp; &nbsp; &nbsp;
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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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