Good day,
If protein expression in blood monocytes was low (detected by western blot) and protein level in serum (detected by ELISA) was high? what dose it mean?
Note: the protein should not leave the nucleus because it’s a DNA-binding protein.
Mirna K. Faiq means the protein is synthesized in monocytes but they are secreted out that's why you found a large amount in ELISA.
Seema Bharatiya
Thanks dear doctor,
but this protein is DNA- binding protein and should not leave the nucleus.
this mean that it’s not necessary that when western blot give result, the ELISA should give the same result ?
Did you perform the ELISA on cell lysates? In that case I would just assume a higher sensitivity of the ELISA compared to WB. Or did you corrected somehow on the different sensitivity?
Silvia Martinelli
Dear doctor, I just measured the protein in serum by sandwich ELISA kit of diabetes patients blood. And the levels were significantly higher than in control healthy group.
when I returned to previous study I found that the protein expression measured by western plot from monocytes of diabetes patients was low.
and this protein not previously measured by ELISA, and normally it should located inside the cell.
so, I was confused, Why its expression is low but its level in serum is high.
Maybe it is better to confirm the protein levels in the cell lysate of monocytes and serum using the same method first (may be ELISA for both). If the result still shows up to be the same as you are seeing now, you can further look into the protein characteristics. For example, the protein might have dual functions, like both as DNA binding and secretory protein. There is also possibility to have a feedback controlling mechanism by secreting out the protein (these are stipulation, and the first thing I would do is to confirm the result using same method).
Agree that just because it's DNA-binding, that doesn't mean it can't be secreted too. But if you are sure that it is not secreted, necrosis-type cell death is the only way I can think of for it to get into serum. Also, are you sure the protein is only expressed in monocytes?
As for your result, are you confident your protocol for making the cell lysate does enough to preserve protein stability? Cell lysates can be fickle in that regard.
Another potential issue are posttranslational modifications. If the secreted form has different PTMs than the intracellular one (which is common), this might affect antibody affinity.
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