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Phospho-protein detection


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#1 science noob

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Excellent

Posted 26 November 2011 - 03:10 AM

Has anyone done phosphorylated protein WBs before? I find that these proteins are less abundant than that of the native form and need a special lysis buffer. Bands have been pretty faint, does that mean that its less abundant?

#2 protolder

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Good

Posted 27 November 2011 - 11:30 PM

yes they are difficult to see , you have to add phosphatase inhibitors to the lysis buffer as Phospho stop of Roche. There are some resins based in IMAC which specifically bond phosphoproteins , but you have to test yours. Actually kinase inhibitors act on the phosphorilation rate of some kinases lowering it , so the determination of inhibition rate is more difficult. Buena suerte

#3 Irene G

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Neutral

Posted 02 December 2011 - 07:21 AM

We don't have any problems using this lysis buffer:
  • 150 mM NaCl
  • 50 mM Tris-HCl pH 7.5
  • 1% Triton X-100
  • 1% NP-40
  • 30 mM NaF
  • 5 mM EDTA pH 8.0
  • 1 mM DTT
  • 0.1 % SDS
  • H2O
You have to add just before using it:
  • 1 mM Na3VO4
  • 2 mM PMSF
  • 10 µg/ml aprotinin
  • 10 µg/ml leupeptin
  • 10 µg/ml pepstatin
Regards
Irene




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