Laboratory Excercises Weeks 9 and 10.pdf

10 Pages
Unlock Document

University of Toronto Scarborough
Biological Sciences
Daman Bawa

Brunt 2011 Laboratoryweek 910 Biochemistry module Analysis of Protein expression in wild type and lactose operon mutant strainsusing SDSPAGE and XgalIPTGOutline Week 9A Induction of Ecoli cells with IPTG and protein sample preparation Week 9B Separation of Ecoli proteins on SDSPAGE and plating cellson XgalIPTG Week10A Analysis of protein expression data with TA and Analysis of growth on XgalIPTG Week 10B Presentation of Group data to the class using powerpointObjectivesUnderstand the techniques that can be used to break opencells and isolate proteinsPreparation of proteins for SDSPAGEPrinciples of SDSPAGEHow todetermine the molecular weight of Proteins using SDSPAGEWhat is a protein expression profileHow to analyze protein data and present it both in oral and written formatsInterpretation of the invivo galactosidase assay using the gratuitous inducer IPTG and the substrate XgalBackground There are severaldifferent biochemicaltechniques that can be used to purify and characterize the macromolecules of a cellFor the purposes of this exercise we will concentration on proteinsProteins have different sizes composition solubility in aqueous solutions as well as other unique physical or chemical properties that can be used to differentiate protein populations In order to study the characteristics or specific proteins in a cell if unicellular or tissue multicellular you must collect the material of interest and lyse the cells to release the proteins In addition you may wish todo subcellular fractionation ofeukaryotic cells to allow isolation ofparticular organelles such asthe nucleus or mitochondria to assay proteins unique to a particular organelleThe choice of the procedure you use will be based on whether you wish to have a total protein population of a subcellular fractionation In addition you must decide if you wish to isolate functional proteins For example if you are only interested in looking at the total protein population by SDSpolyacrylamide gel electrophoresis you may choose to lyse the cells and denature the proteinsin one step this is what you will be doing in this laboratory If you are interested in doing functional assays such as looking at enzymatic activity orproteinprotein interactions then you must lyse the cellsusing a buffer in a manner thatcreates a cell homogenatecontaining proteins that remain in their tertiaryquaternary structure1
More Less

Related notes for BIOB12H3

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.