Isolation of complexes from serum

ComplexescanbeisolatedfromserumbycentrifugationoneitherasucroseorPercollgrADIent.Thesucrosegradientistheeasierofthetwotosetup.

Sucrosegradient:

1. Place5mlof50%sucroseinthebottomofapolyallomer15mlultracentrifugetube.MarkthetopofthislayerwithaMarkerpen.
2. Carefullyload3mlof30%sucroseontothebottomlayerofsucrose
3. Carefullyadd5mlwaterontothe30%sucroselayer
4. Add1mlofserum(containingthecomplexestobeisolated)onthetopofthegradient.Theserumshouldsinktotheinterfaceofthewater/30%sucroselayers.
5. Centrifugeat30000rpmintheultracentifugeusingaspin-outrotorfor15hoursat25⁰Ctopelletcomplexesatbottomoftube
6. Aftercentrifugation,freezethetubesinliquidnitrogenuntilsolid.
7. Usingarazorblade,cutoffthebottomofthetubeapproximatelyacentimeterbelowthemark.
8. Discardtherestofthegradient
9. Allowthesucrosefromthebottomofthetubetothaw,thenpouroff
10. ResUSPendpelletedcomplexesin300mlofwaterorLaemllibuffer(iftoberunonaPAGEgel).

IsolatingcomplexesfromaPercollgradientrequiresthatanoptimalgradientbedeterminedforeachcomplextobeisolated.Forexample,pLL/DNAcomplexes(pLLMw24,000)centrifugeintoaPercolllayerof1.04g/ml.Thebestseparationofserumfromcomplexeswasfoundusingagradientconsistingof2mlsofwater,3mls1.01g/mlPercoll,3mls1.02g/ml,2mls1.03g/ml,1ml1.04g/ml,1ml1.06g/mland1mlof1.1g/ml.Othercomplexesrequireddifferentgradientsforoptimalseparationofcomplexesfromserum.