My protein has ZnF motif, would using of TCEP destroy this and have effect on RNA binding as I need to use it for the Cys labelling with fluorescent dye.
TCEP reduces disulfide bonds. If your zinc finger does not contain disulfide bonds, then it should be safe to use TCEP to keep the Cys residues reduced. The labeling reaction, however, may label a Cys residue that is involved in metal binding, which would destroy that function. On the other hand, those Cys residues may be protected from labeling if the metal is bound.
Dear Aron
as Adam told you, label the cys ia not the best solution for a zinc finger because cys are also involved zinc binding and once that a cys is labbelled it lost its ability to bind zinc. So i dont think that the problem is the tcep but the labbeling. Can you use other labelling strategies? as lys labelling for example?
ciao
Manuele
Thank you Manuele and Adam for the reply. Actually the Cysteine (By replacing Serine 360 which doesn't affect RNA binding) which I would be using for dye tagging is in RRM (RNA Recognition region) (AA 285-371). The ZnF domain is (AA 422-453). But I would need to do some experiments by adding RNA with protein in presence of TCEP so wondering if TCEP had any affects on ZnF binding domain which can be issue here for my experiment? would the cysteine binding with Cy5 can affect RNA binding in this case as well?
Would the cysteine binding with Cy5 can affect RNA binding in this case as well?
It depends on where the introduced Cys residue is relative to the RNA binding site. The Cy5 probe is a pretty large compound, so if the Cys is near the RNA binding site, there is a chance that the Cy5 could get in the way.
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