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 thisenter image description here

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    $\begingroup$ 1) Are you dealing with phosphorylation of tyrosine, threonine, or serine? 2) What is the difference between the control vs. WT? 3) Why do you want to do this — there may be alternative techniques that could be more reliable ... $\endgroup$ – tyersome Feb 9 at 17:49
  • $\begingroup$ Exactly serine to alanine. Fine, actually one allele is knocked out, another allele is mutated. Wt is a parental cell line. control is a single knockout without a mutation. I know there are other methods available but i wanted to verify by western analysis $\endgroup$ – PraveenKumar Feb 9 at 18:04
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    $\begingroup$ Why is there an apparent size difference between the WT and control? $\endgroup$ – tyersome Feb 9 at 19:22
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    $\begingroup$ Did you do SDS-Page or a native gel? $\endgroup$ – Chris Feb 10 at 18:51
  • $\begingroup$ Why is there an apparent size difference between the WT and control? For this Wt i used a different tag (smaller size). Control and my experiment i used the same tag (bigger in size) Did you do SDS-Page or a native gel? SDS- Page $\endgroup$ – PraveenKumar Feb 11 at 9:12

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
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  • $\begingroup$ I agree that observing a shift for a 150 kDa protein may be difficult, but shifts due to phosphorylation can be seen for some proteins on SDS-PAGE gels. See for example this paper. $\endgroup$ – tyersome Feb 10 at 19:45
  • $\begingroup$ @tyersome I guess it is possible in some cases even though it normally shouldn't happen - I edited the answer. Also note that the authors of that paper were "using a modified acrylamide/bisacrylamide ratio" for the gels to get a visible separation of the two bands. $\endgroup$ – Nicolai Feb 11 at 8:42
  • $\begingroup$ Yeah, it will take 15 to 20 hrs to complete the run. sometimes the gel gets a small break. i $\endgroup$ – PraveenKumar Feb 11 at 9:29

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