In cytoskeletal researches concerning the LIMK pathway, I realized that people determines levels of total LIMK2 where as they do determine levels of both pLIMK1 and pLIMK2 with a polyclonal antibody recognizing both phosphorylated forms at the same time. I do not understand why don't they use a phospho specific antibody only against pLIMK2 it the case where they only determine total levels of LIMK2 but not LIMK1.
Any idea?
That is because the antibody that was raised against the antigen can detect both phosphorylation forms (pLIMK1 and pLIMK2) due to the sequence similarity of the site (probably due to being a conserved site)
Muhammad Bakhait Bin Rahmat thank you for your reply. It is plausable that the phospho specific antibody can detect both forms. But to my knowledge, if you determine a phospho form of a protein, then one should detect also its total form in order to understand the ratio. But in the cases i had implied, they do not determine the total form of LIMK1 although they do determine phospho form of that protein. If they do not determine the level of total LIMK1, then why do they need to use an antibody recognizing phospho forms of both LIMK1 and LIMK2 rather then only using an antibody recognizing only the phospho LIMK2 ?
This is beacuse when you raise an antibody against isoform1 and there exist isoform2 having similar sequence/structures in the case of you will inevitably generate antibodies that recognizes both phospho-sites on isoform1 and2. As for total if youre studying isoform1 or isoform2 then you should indeed probe for the specific total isoform -no excuses-. But if youre going to prove for the phospho protein then you can use pLIMK1/2 as usually it is difficult to find a commercially available antibody that can discern the two apart
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