實驗概要
The method provides a protocol for removal of phosphate groups from proteins, before or after blotting. To demonstrate the 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. De-phosphorylated samples should show little or no staining compared to untreated samples.
主要試劑
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
實驗步驟
1. De-phosphorylation of lysates before SDS-PAGE
1) After lysing cells or homogenizing tissue in lysis buffer and determining the protein concentrations, set aside two samples of a lysate/homogenate that is 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 de-phosphorylated sample and one for the control, an untreated sample. We recommend 20–30 μg of protein per lane for crude extracts.
Note: 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. |
2) Spin down the samples. Re-suspend the pellets in the CIP buffer, 1 μg protein per 10 μl buffer with protease inhibitors, EDTA-free.
3) Add 1 unit CIP per microgram of protein to the “ phosphatase” sample. CIP is typically provided as 10,000 units per ml. Incubate up to 60 minutes at 37oC, 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 SDSPAGE. If desired, sodium phosphate (pH 7.4) can be added as a competitive inhibitor, at a final concentration of 10 mM.
2. De-phosphorylation of membranes after transfer
For an antibody that only binds its phospho-protein 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-SDSPAGE. 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.
1) Transfer gel proteins to nitrocellulose or PVDF and block the membrane with 5% BSA in TBS with 0.1% Triton X-100 for one hour at room temperature. Milk contains casein, a phospho-protein which may create background staining if the antibody cross-reacts with the phosphorylated sites.
2) Cut the membrane to obtain a piece containing at least one sample duplicated in the other piece.
3) Place the two pieces in separate containers of the CIP buffer, 3-5 ml per container.
4) Add CIP to the container with the piece to be de-phosphorylated.