For detection of phosphotyrosine level of a particular protein in a cell lysate, there seem to be two options:
Method two depends on there being available an antibody to the pTyr-modified protein and a dual detection system, such as fluorescence at two wavelengths (since the modified and non-modified bands are likely to overlap on the gel.)
I guess that the immunoprecipitation method is more adaptable to different proteins of interest. Other than that, is one method better than the other (eg greater accuracy)? Is normalisation better in method 2 since you can normalise pTyr signal against protein of interest signal?
If the pTyr-modified protein is available, method 2 is straightforward, and the data will be beautiful if you phosph- and total antibodies are from different hosts and you can show signals with different fluor-colors.
If the pTyr-modified protein is not available, you have to choose method 1.
Not really an answer to your question, but I think to get the true picture it may be necessary to interrogate individual cells, or even sub-cellular compartments. This short communication/paper should let you know my concerns, even though the title may seem of topic, the article is more general, even though the main example concerns DNA repair.
https://www.researchgate.net/publication/357484839_Is_DNA_repair_controlled_by_a_biological_logic_circuit
I presented more on this work in Boston Ma. in April at the Cell and Experimental Biology meeting, this Powerpoint gives the wider biological implications of this type of regulation in rapid (sub second) decision making.
https://www.researchgate.net/profile/Philip-Penketh/publication/360149650_Phosphologic_in_DNA_repair_and_rapid_cellular_data_processing_CEB-2022/links/626982d905d79a3968a5fcb9/Phosphologic-in-DNA-repair-and-rapid-cellular-data-processing-CEB-2022.pdf?_sg%5B0%5D=6NRY4XF6eiV1irthYkGC06IExxt5Kx6MriH1RXc0EUwqQ-Cq9364cIlm8ac6VdhWsoPrm07Nrw92k1L9-wLJHA.04pzWPOBeElef7ehL9kFBPJeprlyMqDEe_vdhBx-AJA2Vo8OzY9xsIPNDIXUfv5TJ6XOaPES-D1XR6xRrDU6nw&_sg%5B1%5D=dJEQkjAjPiPS2jIBuFfzQDtbHYhN9HeVq6pvV-ZbZK7l7OFAkZzmZQslPKCbewYp_Qn5h49Dr3k6STW4BN9-K-yCm-LCKzgYjO3XNgysBSes.04pzWPOBeElef7ehL9kFBPJeprlyMqDEe_vdhBx-AJA2Vo8OzY9xsIPNDIXUfv5TJ6XOaPES-D1XR6xRrDU6nw&_iepl=
All the best, Phil
If you're after pTyr, there are virtually no antibodies which are specific for certain motifs (in contrast to pSer and pThr, see e.g. these phosphorylations of PKB/Akt). So you'll have to identify or pre-purify your protein of interest (2D electrophoresis/WB, possibly in combination with MS analysis) might be an option, if standard PAGE and/or IP cannot resolve your protein well.
The famous PY20 monoclonal anti pTyr might be a good antibody to begin with.
