Hi

I am doing InVitro Ubiquitination assay because my major is phytochorme biology.....phytochromeA is ubiquitinated by one of the repressor protein COP1.this COP1 working as E3 Ligase (contain Zinc finger motif).so basically we are using purified COP1 as E3 ligase and adding our purified PhytochromeA...

we are initiating the experiment with adding 10 ug of Ubiquitin for 40 ul total reaction...we are adding E1 and E2 (from Boston biochem.final is 5 to 20 ng each) and E3 (our COP1) 0.5 to 1 ug..(finally E3COP1 and phytochormeA ratio is 2:1 or 3:1) 100mM Tris-cl pH 7.45-10 mM ATP and 5-10 mM MgCl2..0.5 to 1 mM DTT.and our COP1 need ZnCl2 (because this COP1 contain zinc finger motif) so 20 to 50 uM ZnCl2..this is reaction condition (total 40 ul) but we are not getting any polyubiquitination bands with PhyA ...we are using some times GST Ubi also for western detection.. please give some suggestionbecause its proved that COP1 act as E3 ligase.we need to change any E1 or E2 concentrations (now each 10 to 20 ng ) or E3 ligase is 500 ng to 1ug (is it Ok?) or energy system problem .. we are not getting key point...


please clarify...thank you in advance..