Hello
Today i want to ask very simple but seemingly important question.
When conducting western blot, i have to see the expression of phospho form of protein(Protein of my interest). To measure exact expression of phsopho form of the protein, i see both the phospho form and also total form.
When using related antibodies, antibody specific for phospho form gives me
vivid information that it detects phospho form of protein but in case of total protein, is it right to use antibody of the name of protein itself?
I want to see the nucleus translocation of the protein by immunocytochemistry
and during the experiment, i want to make sure about what i asked above.
Thank you and always appreciate for all support i receive in researchgate
I wonder if the phospho form and the non phospho form could be separated by native electrophoresis (without SDS)? If it was the case, the transfer of the proteins for immunodetection as a "normal ,western blot" might allow to detect the phospho and non phospho form by the use of different antibodies.
Run duplicates on the same gel, cut, stain one set for total protein and the other side for phospho protein. Don't use PBS for phospho. Also. use filtered BSA for the blocker. Make sure you have a loading control to normalize to. Or make a stand curve with known amount of protein on the same gel you are dividing into 2 blots. See how the stats come out :)
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