I want to see the phosphorylation shift in my protein of interest. I have created a point mutation in my protein. so that it will not able to go for the phosphorylation compare to my control. i want to check by western analysis. i can see a very small shift. is it possible to get a better shift? Another important thing is my protein size is ~150 kDa. can you please share any suggestions on this
The run length of bands in an SDS-PAGE (which your western is probably based on) depends - at least in theory - only on the size of the protein: SDS binds to proteins with a fixed size ratio and thereby provides a negative charge based only on size. Unless you have reason to believe that your protein is an exception from the usual (which might be indicated by things like not running at the expected size) you will very likely not be able to see a band shift (change in run length) based on presence or absence of phosphorylation in a standard western blot.
Things you could try to detect the phosphorylation:
- Use phospho-specific antibodies. These are often expensive, tricky to use or just not available, but this is the only ting I can think of working with a standard western blot setup
- run a native-PAGE, that way your run length depends on shape, size & charge of the protein. I still wouldn't expect a single phosphorylation to be cause a visible shift for a 150kDa protein, but you can try. This might require using longer run-time of the gel to get really high resolution for the size you are looking for