26/04/2017 Crystallographyforbeginnerspart4settingupyourfirstcrystallizationexperiment|Benchwise

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Crystallographyforbeginnerspart4settingupyourfirst
crystallizationexperiment

PostedbyChenGumanon17/11/2012inCrystallography,Protein,Proteincrystallization,Tips,youtube
Youhavepuriedyourproteinandyourereadyforyourrstproteincrystallizationexperiment.InthispostIwillgiveyoustepbystep
instructionshowtosetyourrstproteincrystallizationexperiment.

Lysozyme crystal growth

Materialsandequipmentlist

Beforeyoubegin,verifyyouhavetheresourcesrequiredforproteincrystallizationexperiment:

Temperatureandhumidcontrolledrefrigerator/chamber/roomforcrystallizationplateincubation.Makesureyouhavethemsetto
20Cand4C(optional).
Yourproteinatconcentrationof~300600M(1020mg/mlfora30kDaprotein).
AcommercialsparsematrixscreeningkitsuchasissoldbyHamptonResearch(hp://hamptonresearch.com/menus.aspx?
id=2&cid=1),Moleculardimensions(hp://www.moleculardimensions.com/shopdisplaycategories.asp?
id=1&cat=Crystallization+Screens),Emeraldbiosciences(hp://www.emeraldbiosystems.com/c1crystallographyscreens.aspx)etc.
Orderdeepwellblock(hp://www.eppendorf.com/int/index.php?l=1&action=products&catalognode=28400)andtransfereach
conditionofthematrixtoanewwellintheblock.Sealandkeepat4C.
Lookingatthelistsofkits,youmustaskyourselfwhichonetoorder?Well,youllwantthescreenwhichwillsparselysampleas
muchaspossibledierentcrystallizingprecipitant(salts,organicandinorganicsolvents,PEGSetc.)whilevaryingtheconditions
slightlyforeverytypeofprecipitant.Somenamesthatwehavebeenusinginthelab:Index,Crystalscreen1+2,Wizard,JCSG,
structureetc.
96wellsiingdropcrystallizationplate(MRC)suchasthisone(hp://www.moleculardimensions.com/shopdisplayproducts.asp?
id=50&cat=The+MRC+Crystallization+Plate+).
Awellcalibratedsetof0.12l,0.510land10200lpipeors+tips.
Amultichannelof10100.
Crystallographysealingtapesuchashere(hp://www.jenabioscience.com/cms/en/1/catalog/287_sealing_tape.html)andregular
standardwidthocetapesuchas3MofScotch(Ipersonallyndthematenishofthe3Mtapetobejustattherightbinding
capabilities,nottoolightandnottoostrong)

Seingupthecrystallizationplate

1.Takeanew96wellsiingdropplateandwriteonitssidethefollowinginformation:

Date
Yourname(andlabiftheincubatorservesotherlabsaswell).
Proteinnameandconcentrationattheupperandlowerwells(useupanddownarrows).
Buercompositionoftheproteinsolution.
Nameofthesparsematrix.
Incubationtemperature.

2.Prepareyourproteinsolutionatx1andatx0.5concentrations.
3.Usingthemultichannel,copy5080micoliersofthematrixstockontothenarrowwellofeachcolumn/row(reservoirwell).
4.Usinganaluminumfoilcovertheplateexceptforcolumn1.

5.Usethe0.12microlierpipeetodrawhalfamicrolierfromthelowerconcentrationproteinsolutionanddispenseitontothetop
https://benchwise.wordpress.com/2012/11/17/crystallographyforbeginnerspart4settingupyourfirstcrystallizationexperiment/ 1/2
26/04/2017 Crystallographyforbeginnerspart4settingupyourfirstcrystallizationexperiment|Benchwise
5.Usethe0.12microlierpipeetodrawhalfamicrolierfromthelowerconcentrationproteinsolutionanddispenseitontothetop
well.
Toavoidinsertingairbubblespushthepipeebuontotheitsrststoppingpoint.
6.Withthesametipdrawhalfamicrolierfromthehigherconcentrationsolutionandplaceitinthelowerwell.
7.Replacetheusedtipwithanewoneanddrawhalfamicrolierfromthereservoirwellanddispenseitwithoutmixingontotheupper
drop.Dothesameforthelowerwell.
8.RepeatthesameoperationforallrowsBH;doitasfastasyoucansincetheexperimentofthedispenseddropsisrunningandtheopen
airdehydratesthewaterothedrops.
9.Cutoaplatewidthstripoftheocetapeandcarefullysealtherstcolumn.
10.Repeatthisstepforalltherestofthecolumn.
11.Whennishing,removetheocetapeandquicklyaspossibleresealtheplatewithcrystallographygradesealingtape.Makesurethe
sealiscompletealongtherimsofthewellsandthatnoaircanpassbetweenthewells.
12.Placetheplateattheincubatorandmonitorforcrystalgrowth.Howdoyoudothat?Seemynextpostonmonitoringandevaluation
ofcrystallizationexperiments.

SearchingfortheSDSPAGEimageofyourprotein?YoumightwanttoconsiderLabguru(hp://www.labguru.com/tour),awebbasedlaboratory
managementsystem.Goahead,itsfree(hp://www.labguru.com/)!

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