In my opinion, it is only necessary to overexpress the ligand (or inject recombinant ligand) if detection of endogenous receptor phosphorylation is impossible with standard techniques. Otherwise you are adding unnecessary variables and complicating interpretation of your results. See if you can detect the phosphoreceptor first by whatever method you're most comfortable with.

If you do IHC, can you not just colocalize the phosphoreceptor with a cell-type specific surface antigen? Depending on how you permeabilize your tissue, you can limit detection to cell surface antigens only. EM or array tomography would be ideal here.

If you do WB, fractionate the tissue to isolate membrane and cytosolic compartments. Probe for both the phosphoepitope of the receptor and the native protein (unphosphorylated) in both fractions. Phosphorylation of certain RTKs mediates their internalization so you could get quite a bit of information here. A kit is not needed for this and there are much better alternatives to NP-40.

Instead of subcellular fractionation, you can IP with and antibody against the standard RTK and IB with antibodies against phosphoepitopes (I.E...IP: TrKB, IB: Phosphoserine, phosphothreonine, etc). Be careful with selection of tissue homogenization buffer (don't use RIPA) and IP buffers (STEN is a good starting point).

Good luck Benoit.

Dear Grant,

Thank you very much for your kind and helpful answer.

EM and array tomography would be surely ideal ... but I have no access to such tecnology for now.

IHC could be great but quantification of pharmacological effects based on phosphorylated receptor vs total receptor in costaining is tight.

About WB, IP could be great, but we need to first of all find THE good antibody.

Finally, we will manage both IHC and WB ... focusing in first on the Ab validation from fractionated proteins I think. You mentioned some much bette alternativs to NP-40, could you please precise your mind ?

My target protein is heavy (>100kDa), any advice to improve migration and transfer on membrane ?

Nice day

Benoit

Sure. As far as the buffer goes, I have had good luck with 1% CHAPS buffer for solubilizing proteins:

3 ml  1 M Tris-Cl pH 7.5

3 ml  5 M NaCl

10 ml  10% CHAPS

84 ml  ddH2O

Its very efficient for solubilizing membrane proteins (your receptor). Also, because it is nondenaturing, all your phosphoepitopes should be conserved. IP with CHAPS buffer is very common as it maintains protein complexes/structures.

For transfer of large proteins (100kDa isn't really that large though), I'd recommend wet transfer with ~20% methanol. In general, it depends on protein hydrophobicity here. The more hydrophobic the protien, the harder it is to transfer. Also, if you use nitrocellulose, pick a product with a larger pore size (.45um should be fine). I have successfully transferred proteins up to 200kDa using a semi-dry system with the following buffer:

200ml Methanol

800ml ddH20

3.03g Tris

14.4g Glycine

Good luck.

Dear Grant

It's great to read from you. Many thanks for your advices, and we will try the 1% CHAPS

For transfert of my protein (190kDa) I use 20% methanol buffer, but I wonder if a lower methanol percentage would not fit better as methanol can inhibit the large proteins transfer.

Have a nice day

Benoit