2 replies to this topic

[science noob]

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?

[protolder]

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

[Irene G]

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