Hi all,
I am looking at cytoplasmic mislocalization of a mainly nuclear expressed protein. Therefore I performed a nuclear cytoplasmic fractionation, which worked perfectly (no leakage from nucleus in cytoplasm or the other way around). The problem is that my protein of interest has a totally different molecular weight in the cytoplasm (+- 40kDa) than in the nucleus (+- 65kDa = expected MW). I tested four different antibodies, but each show similar results. Even when I overexpose the nucleus to see a potentially faint band in the cytoplasm at the same height, I don't see anything. Do I quantify the bands that appear at a lower MW in the cytoplasm? Or how do I solve this and know for sure that these bands are the right protein.
Immunofluorescent imaging of this protein shows a partial mislocalization, so I know that the protein is present in the cytoplasm.
Can somebody please help me with this?
Thank you!
Dear Elke,
Can you inform which protein you are assessing? The formation os certain complexes may happen during translocation to the nucleus, this is rather common. Perhaps your translocated protein is associated with another small protein, and when you avail it by immunoblot you still verify its presence, nevertheless the complex migrates less in the gel.
Eduardo.
Hi Eduardo,
The protein I’m looking at is hnRNPK, a heterogenous ribonucleoprotein which acts as a splicing factor and transcriptional regulator in the nucleus. A
Hi Elke,
I think you can check if DTT is contained in loading buffer. DTT can change the weight of protein has -SH.
Another question : Which protocol you used for nuclear cytoplasmic fractionation? I couldn't fractionate nuclear and cytoplasmic completely. I used Histone H3 for nucleus marker, GAPDH for cytoplasm marker.
Thank you very much.
Elke Braems
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