Lab.2.

PROTEINASSAY
Thepurposeofthislaboratorytechniqueistodeterminetheamountofproteinpresentinasample.Thesamplemightbe
blood,atissuehomogenate,orproteinssecretedfrombacteria.Orthesamplecouldbeasampleoffoodorbeverage,to
verifytheproteincontentclaimedonthelabel.Inthiscaseweareanalyzingsamplesof,albumin,specificallyBovineSerum
Albumin(BSA).BSAisthemostabundantproteininserum(blood,minusredandwhitecells).Assignment:Whatarethe
functionsofthisprotein?Whatisthesizeofthisproteinmolecularweight,numberofaminoacids?Other
interestingfactsaboutalbumin?Also,obtainthisinformationforactinandmyosinproteins.Bepreparedtoreport
yourfindingsnextclass.
WewillbeusingtheBioRadQuickStart(Bradford)reagent.ThisreagentcontainsCoomassieBlue,adyethespecifically
bindstoproteinmoleculesandchangescolor.Thesamedyeisusedtostainproteinsingels.Theintensityofthecoloris
relatedtotheamountofprotein.Thusyoucanmixknownamountsofprotein(e.g.BSA)withthedyereagentandmeasure
theintensityofthecolorinaSpectrophotometerandthencomparetheintensityfromanunknownsampletoestimateits
proteinconcentration.http://www.piercenet.com/products/browse.cfm?fldID=02020105
Materials.Eachstudentneeds
4glasstubes
~3mlofBioRadQuickStart(Bradford)reagentor10mlforateamof4.
~100uloftheBovineSerumAlbumin(BSA)standardsolution,1ug/10ul(=1mg/10ml).
DistilledWaterina15mltube.
One,1mlplasticcuvette
Eachteamneedsatuberack,andaspectrophotometer.
Methods.Eachstudentshouldmakeatable(Table1)intheirnotebookthatshowsthevolumesofBSA,waterandBioRad
Reagentthatwillbeaddedtoeachtubeandindicatetheamountofprotein(ugineachtube).Tubesshouldbepreparedfor
eachstandardamountofBSA:0,10,20and50ulplusdistilledwatertobringthevolumeineachtubeupto500ul.Also,
preparetubestwodifferentamountsofyourunknownproteinsamplepluswaterto500ul.Datethepage.Ifthesesample
volumesdonotyieldAbsorbancevalueswithintherangeoftheBSAstandardsyoushouldtrydifferentamounts.
Onceallofthetubes,standardsandunknowns,havebeenpreparedadd500uloftheBioRadreagenttoeachtube.Afterthis
additionthetotalvolumeineachtube,standardsandunknowns,shouldbe1000ul.Asyouaddthedyemixeachtube
severaltimessothatthecolorisuniform.
SetupthespectrophotometertoreadthelightAbsorbanceofeachsample.CalibratethespectrophotometerwithdH 2O.
Makesurethatthecuvetteisorientedsothatthelightpathpassesthroughtheclearwindowsofthecuvette.Setthe
wavelengthto595nm.Pouroutthewater,pourinyourstandard0intothecuvetteandwritedowntheAbsorbancereading
intheTableIyournotebook.Pourthe0standardbackintoitstubeandrepeattheprocesswitheachoftheotherstandards
andunknowns,recordingthereadingsinTable1.
Resultsastheyshouldappearinyournotebook:
A.TableIshowingtheulwater,ulBSA,BSA,ulunkown,ugproteininunknownandtheAbsorbancereadingsfor
everyoneinyourgroup.
B.Plotallofthestandardvaluesforyourgroup(notaverages)withtheugBSAvaluesonthexaxisandtheAbsorbance
(A695)ontheyaxis.Drawalineorcurvethatbestfitsthepoints.Ifyourstandardpointsdonotdescribeaclearlineorcurve
repeattheMethods.IftheA695readingsforyourunknownsaregreaterthanyourstandardreadingsrepeattheunknownwith
asmalleramountofsampleorwithadilutedsample.Orifthereadingsarelowuseagreateramountoftheunknownsample.
Keepexactnotesonwhatvolumesyoupipettetomakethedilutions.
Challengequestion:whatisthemolarconcentration(Mmoles/liter)ofBSAinthestandardsolutionandhowmanywater
moleculesarethereperBSAmoleculeinthatsolution?

C.Concludingstatements:Describeyourobservationsanduncertainties.Giveanestimatefortheug/ulproteininyour
unknown.Suggestwhatyoucoulddotoimproveyourmeasurementsandresults.
Show this to your lab instructor before leaving today.