We are expressing a protein by transient tranfection in Hek 293 cells. When performing western blot analysis against our protein of interest (PAH), we obtain two bands, being, probably, our protein phosphorylated and dephosphorylated. We would want to confirm this hypothesis and we have thought in many options( inmunoprecipitation and MALDI analysis, 2D and proQdiamond, an especific antibody against our protein phosphorilated-non comercially aviable-...) and also incubate with some phosphatase and analyze again by western blot. If anyone knows a protocol we would be very grateful.
Protein dephosphorylation protocol (abcam)
Removal of phosphate groups from proteins, before or after blotting.
Print this protocol.
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.
Materials
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
I also used a protocol similar to the one suggested by Stefano. More in detail, after cell lysis I treated one mg of total crude extract (I had to perform a GST-pulldown afterwards) with 200 units of CIP in CIP buffer (50 mM Potassium Acetate, 20 mM Tris-Acetate, 10 mM Magnesium Acetate) at 37°C for 1 hour. I did not add BSA in the buffer (as requested in the recipe) since this would cover my bands of interest...
Juan, I suggest including appropriate controls for your dephosphorylation experiment as well to conclude that it is phosphorylation that you are dealing with. I do at least four separate reactions for in vitro dephosphorylation: +Ppase -Pphase inhibitor; +Ppase +Ppase inhibitor; -Ppase +Ppase inhibitor; -Ppase - Ppase inhibitor.
Good luck!
Protein dephosphorylation protocol (abcam)
Removal of phosphate groups from proteins, before or after blotting.
Print this protocol.
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.
Materials
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
Use protein in bulk quantities: Do Phosphatase treatment and try free phosphate analysis by Fisk and Subbarow method.
Lamda phosphatase from NEB gives you the detailed protocol for cell lysate dephosphorylation. It works well with whole cell extracts as well as IP proteins as well as the recombinant proteins. Follow the protocol exactly as mentioned in manufactures instruction page.
Hi,
Firstly, I would like to ask you whether the antibody you are probing is phospho antibody or protein specific antibody.
If the protein specific antibody is showing up as two bands, then I would say that the bands were partially degraded or digested or isomers but not the phosphorylated or dephosphorylated ones. The reason being that the phospho or dephospho only adds 80 or 90 kDa mol wt. to protein that is hardly seen as a shift in conventional gels.
I would suggest you to please do maldi or phosphostain or phospho antibody to confirm the same..
Hope you understand.
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