Materials

MiliporefiltertypeHA0.45micron

Cultureplates(Linbromodel76-033-05)

ProteininDDWorHEPESbuffer(10mg/mL)

Vacuumgrease

Syringewith18Gneedle

Procedure

1.	Milliporefilterallsolutionstobeused.

2.	Dialyzeproteinfor24hoursagainstwater,ifpossIBLe.Otherwise,includeminimumrequirementsforkeepingtheproteinsoluble.IfbufferisnecessaryuseHEPESbuffer.

3.	Fortheinitialscreeningusethebuffersandprecipitantsrecommendedinthefastcrystalscreen(Jancarik,J.&Kim,S.H.1991J.Appl.Cryst.24,409),asinstructed,andthehangingdropmethodwiththeLinbrotissuecultureplatesthathave24wells,1.7cmdiameter,1.6cmdeep.Seediagram.

4.	Useaproteinsolutionwithastartingconcentrationof10mg/mL.Thisconcentrationmayhavetobeadjusteddependingonthenumberofthedropsthatprecipitate.Theconcentrationisaboutcorrectwhenone-thirdtoone-halfofthedropsdevelopprecipitatewithinoneweek.

5.	Deposit0.7mLofreservoirsolutionintothewell.Combine2microlitersoftheproteinsolutionand2microlitersofthereservoirsolutiontomakeuptheproteindroplet.

6.	Incubateoneatroomtemperatureandoneatcold(4-10°C)temperature.

Notes

1.	CrstallizationscreeningkitsandothersuppliesareavailablefromHamptonresearch.

TakenfrommethodsbyJ.JancarikandS.H.Kim

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