LareScaleCellLysis
- MakefreshcoldLysisBuffer
LysisBuffershouldbeabufferinwhichthetargetproteinissoluble.
TheLysisBuffershouldbecompatIBLewithdownstreampurificationprocesses,e.g.minimalamountofEDTAorDTTifNicolumnwillbeused.
HereisanexampleofLysisBuffer
25mMTris-HCl,pH8.0
500mMNaCl
14mMbeta-mercaptoethanol
Detergentcanbeincludedforlesssolubleproteinsorwhenproteinsolubilityisunknown.1%TritonX-100hasnoeffectonTurboNucleaseactivity.
TurboNucleasehasthesameactivityin150mMNaClor500mMNaCland400mMimidazole.
- ResUSPendthawedcellpasteinLysisBuffer
Use2-10mlLysisBufferforeachgramofcellpaste.TurboNucleasecanreducetheamountofLysisBufferused.
Weroutinelyuse2mloflysisbufferforeachgramofcellpellets. - AddTurboNucleaseto25unit/ml
Proteaseinhibitorscanbeaddedatthesametime.
IfthelysisbuffercontainsEDTAorEGTA,add10-foldmoreTurboNuclease. - Lyzecellsbymechanicalorchemicalmethodsoniceoratroomtemperature
TurboNucleasealsoreducestheviscosityoflysatelyzedbymicrofluidizer. - Clearlysatebycentrifugationforcolumnloading
Thereducedviscositymakesitpossibletoclearthelysateatlowerspeed.35,000g(~16,000rpm)for1hourissufficient.
Lysatecanbeloadedto"Crude"columnswithoutclearance.
ParallelLysisofMultipleInsectCellSamples
- Freezecellspelletsof5-10mlcultureondryicebriefly
Freezeandthawfacilitateslysis. - Thawthefrozenpelletsandcompletelyresuspendin~1mlLysisBufferwithTurboNuclease
- Transferthecellsuspensiontoamicrotubeandsitthetubesonafloaterrack
- LyzecellsusinganUltrasonicCleanerwithicewaterbathfor10min
UltrasonicCleaner(manychemistsuseit)ismuchcheaperthanprobesonicator.ItcostsafewhundredsUSdollarsforanewmodelandlessthan$100forausedoneorajewelrycleanerfromaconsumergoodsstore.UltrasonicCleanerismuchbetterandcheaperthanthosefancymulti-probesonicatorswiththefollowingadvantages.
Thereisnocross-contaminationsinceeachsampleisenclosedinamicrotube.
Thesamplesarealwayscoldaslongasiceisaddedinthewater-bath.
Thereisnolimitonthenumberofsamplesprocessedinparallel.AsmallUltrasonicCleanercaneasilyhold48samples
Thelysatecanbeusedforanalysesofproteinexpressionofwholecelllysate,solublelysate,oraffinitypull-down.
EtonBioLysisBuffer
25mMTris-HCl,pH8.0
500mMNaCl
20mMImidazole,pH8.0
14mMbeta-mercaptoethanol
0.5%TritonX-100
25units/mlTurboNuclease
MaterialSafetyDataSheet
Date:Oct26,2015
1.PRODUCTANDCOMPANYIDENTIFICATION
1.1Productidentifier
Productname:TurboNuclease
CatalogNo:1400010050,1400010100,1400010250
1.2Relevantidentifiedusesofthesubstanceormixtureandusesadvisedagainst
Forresearchuseonly.
1.3Detailsofthesupplierofthesafetydatasheet
TollFree:1-800-758-1630
Tel:1-800-758-1630
Fax:1-800-507-2912
1.4Emergencytelephonenumber
1-800-758-1630
2.HAZARDSIDENTIFICATION
2.1Classificationofthesubstanceormixture
Notahazardoussubstanceormixture
2.2GHSLabelelements,includingprecautionarystatements
Notahazardoussubstanceormixture
2.3Otherhazards
None
3.COMPOSITION/INFORMATIONONINGREDIENTS
3.1Substances
Synonyms:RecombinantSerratiamarcescensextracellularendonuclease
CAS-No | EC-No. | Index-No | Classification | Concentration |
Water |
7732-18-5 | 231-791-2 | - | - | >=45% |
Glycerol |
|
|
|
|
56-81-5 | 200-289-5 | - | - | 50% |
Tris(hydroxymethyl)aminomethanefreebase |
77-86-1 | 201-064-4 | - | Xi,R36/37/38 | 0.6% |
Enzyme |
- | - | - | - | <=0.025% |
Magnesiumchloridehexahydrate |
7791-18-6 | 232-094-6 | - | - | 0.1% |
Sodiumchloride |
7647-14-5 | 231-598-3 | - | - | 0.3% |
ForthefulltextoftheR-phrasesmentionedinthisSection,seeSection16.
4.FIRSTAIDMEASURES
4.1Descriptionoffirstaidmeasures
Eyecontact
Checkforandremovecontactlensesandflushwithcopiousamountsofwater;assureadequateflushingbyseparatingtheeyelidswithfingers;callaphysician
SkinContact
Flushwithcopiousamountsofwater;removecontaminatedclothingandshoes;callaphysician
Inhalation
Removetofreshair.Ifnotbreathinggiveartificialrespiration
Ingestion
Ifswallowed,nevergiveanythingbymouthtoanunconsciousperson.Rinsemouthwithwater.
4.2Mostimportantsymptomsandeffects,bothacuteanddelayed
Noinformationavailable
4.3Indicationofanyimmediatemedicalattentionandspecialtreatmentneeded.
Noinformationavailable
5.FIREFIGHTINGMEASURES
5.1Extinguishingmedia
Suitableextinguishingmedia
Waterspray,alcohol-resistantfoam,drychemicalorcarbondioxide
5.2Specialhazardsarisingfromthesubstanceormixture
Noinformationavailable
5.3Adviceforfirefighters
Wearself-containedbreathingapparatusandprotectiveclothingtopreventcontactwithskinandeyes.
6.ACCIDENTALRELEASEMEASURES
6.1Personalprecautions,protectiveequipmentandemergencyprocedures
Usefullpersonalprotectiveequipment.Evacuatepersonneltosafeareas.Avoidbreathingvapors,mistorgas.
6.2Environmentalprecautions
Preventfurtherleakageorspillage.Preventproductfromenteringdrain.
6.3Methodsandmaterialforcontainmentandcleaningup
Keepinsuitable,closedcontainersfordisposal
7.HANDLINGANDSTORAGE
7.1Precautionsforsafehandling
Avoidskin/eyecontact.Useprotectiveequipmentasneeded.Washcontaminatedclothingbeforereuse.
7.2Conditionsforsafestorage,includinganyincompatibilities
Keepcontainertightlyclosed.Storeat-80?C.
7.3Specificenduse(s)
Nodataavailable.
8.EXPOSURECONTROLS/PERSONALINFORMATION
8.1ControlParameters
Thisproductcontainsnohazardousmaterialswithoccupationalexposurelimits.
8.2Exposurecontrols
Engineeringcontrols
Provideshowerandeyewashstation
Personalprotectiveequipment
EyeProtection | Wearsafetygoggles |
SkinProtection | Wearprotectiveclothing |
HandProtection | Wearprotectivegloves |
Respiratoryprotection | WearNIOSH/MSHAapprovedrespirators |
9.PHYSICALANDCHEMICALPROPERTIES
9.1Informationonbasicphysicalandchemicalproperties
Appearance | Clear,colorlessliquid |
pH | 8.0 |
Meltingpoint | Nodataavailable |
Flashpoint | Nodataavailable |
Boilingpoint | Nodataavailable |
Ignitiontemperature | Nodataavailable |
Lowerexplosionlimit | Nodataavailable |
Upperexplosionlimit | Nodataavailable |
Watersolubility | Soluble |
9.2Othersafetyinformation
Nodataavailable.
10.STABILITYANDREACTIVITY 10.1Reactivity
Nodataavailable
10.2Chemicalstability
Stableunderrecommendedstorageconditions.
10.3Possibilityofhazardousreactions
Nodataavailable.
10.4Conditionstoavoid
Nodataavailable
10.5Incompatiblematerials
Strongoxidizingagents
10.6Hazardousdecompositionproducts
Hazardousdecompositionproductsformedunderfireconditions:carbonoxides
11.TOXICOLOGICALINFORMATION
11.1Informationontoxicologicaleffects
Acutetoxicity
Nodataavailable
Irritationandcorrosion
Nodataavailable
Sensitisation
Prolongedorrepeatedexposuremaycauseallergicreactionsincertainsensitiveindividuals
Chronicexposure
IARC:Nocomponentofthisproductpresentatlevelsgreaterthanorequalto0.1%isidentifiedasprobable,possibleorconfirmedhumancarcinogenbyIARC.
SignsandSymptomsoExposure
Prolongedorrepeatedexposurecancause:Nausea,Headache,Vomiting
PotentialHealthEffects
Inhalation | Maybeharmfulifinhaled.Maycauserespiratorytractirritation |
Skin | Maybeharmfulifabsorbedthroughskin.Maycauseskinirritation |
Eyes | Maycauseeyeirritation |
Ingestion | Maybeharmfulifswallowed. |
Targetorgans | Kidney |
12.ECOLOGICALINFORMATION
12.1Toxicity
Avoidreleaseintoenvironment
12.2Persistenceanddegradability
Nodataavailable
12.3.Bioaccumulativepotential
Nodataavailable
12.4Mobilityinsoil
Nodataavailable
12.5ResultsofPBTandvPvBassessment
Nodataavailable
12.6Otheradverseeffects
