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
Dephosphorylation of lysates before SDS-PAGE
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.
If possible, avoid using sodium orthovanadate (a component of RIPA lysis buffer) and EDTA in the lysis buffer: 10 mM sodium orthovanadate inhibits CIP activity (10 units) by 90% and 50 mM EDTA inactivates CIP (10 units) almost 100%. It is essential if using crude extracts that protease inhibitors are included in the CIP buffer.
Resuspend protein/lysate in the CIP buffer, 1 µg protein per 10 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 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.
Dephosphorylation of membranes after transfer
For an antibody that only binds its phosphoprotein target when the protein is denatured, treating the membrane with phosphatase post-transfer may be preferable to treating the non-denatured lysate with phosphatase, pre-SDS-PAGE. For a well-controlled comparison, the membrane treated with phosphatase should be a piece cut from a blot of a single gel containing a duplicate lane or lanes.
Transfer gel proteins to nitrocellulose or PVDF and block the membrane with 5% BSA in TBS with 0.1% Triton X-100 for 1 hr at room temperature. Milk contains casein, a phosphoprotein which may create background staining if the antibody cross-reacts with the phosphorylated sites.
Cut the membrane to obtain a piece containing at least one sample duplicated in the other piece.
Place the two pieces in separate containers of the CIP buffer, 3-5 ml per container.
Add CIP to the container with the piece to be dephosphorylated.