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Protein dephosphorylation protocol

Removal of phosphate groups from proteins, before or after blotting.
Last edited Sun 21 Mar 2021

To demonstrate specificity of an antibody for a protein in its phosphorylated state, proteins can be dephosphorylated either before SDS-PAGE or after transfer to a membrane. Dephosphorylated samples should show little or no staining compared with untreated samples.


  • Calf intestinal alkaline phosphatase (CIP)
  • CIP buffer: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH to 7.9 at 25ºC
  • ​Protease inhibitor cocktail, EDTA-free

Stage 1 - Procedure



After lysing cells or homogenizing tissue in lysis buffer and determining the protein concentrations, set aside two samples of a lysate/homogenate that are expected to be positive for the phosphoprotein.

  • Typically, only two lanes of a gel are needed to demonstrate the specificity of a phosphoprotein-specific antibody: one for the dephosphorylated sample and one for the control, an untreated sample. 
  • We recommend 20-30 µg of protein per lane for crude extracts.

Resuspend protein/lysate in the CIP buffer, 1 µg protein per 1 µL 1X buffer with protease inhibitors, EDTA-free.


Add 1 unit CIP per µg of protein to the "+phosphatase" sample.

  • CIP is typically provided as 10,000 units per ml. Incubate for up to 60 min at 37°C, although shorter times and a lower temperature may be effective if proteolytic degradation is a concern. 
  • Samples can be frozen and stored at this point or processed as usual for SDS-PAGE. 
  • If desired, sodium phosphate (pH 7.4) can be added as a competitive inhibitor at a final concentration of 10 mM.