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L17_SDS Page.pdf - 10/29/2021 Mandatory Masking |...

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10/29/20211Mandatory Masking | Quarantine PeriodANNOUNCEMENT | COVID-19 Positive Case ConfirmedFor the health and safety of your peers and the OU community, maskswill be required while this class meets in person and will continue ineffect until the quarantine period,November 10.If you do not have a mask, forgot your mask, or have an exemption frommasking, please see me. Please take care of this in the next threeminutes before class begins.The plasmid containingadhPwas transformed intoBL21DE3cells, and grown withAmpicillincontaining mediumAfter reaching an OD600 of ~0.6, protein was overexpressed bythe addition of1 mM IPTGThe resultant AdhP protein will have aC-terHis Tagwhichwill facilitatepurificationof the protein usingNi-NTAaffinitychromatographyAlso, has a C-terV5-epitopewill enabledetectionof theprotein usingWestern-blotAdhP Expression in pET101D vectoradhP12
10/29/202121)Lyse the cells using (detergent + lysozyme)2)Use Ni-NTA chromatography to purify the proteinSteps involved in the purification of AdhPResuspension Buffer:20 mM sodium phosphate, 300 mM NaCl,5/10 mM Imidazole, pH 8(pI of AdhP: ~6)Wash buffer:wash unbound proteins (20 mM sodium phosphate,300 mM NaCl,30 mM Imidazole, pH 8)Elution Buffer:Elute with high concentration of imidazole (20 mMsodium phosphate, 300 mM NaCl, pH 8 with250 mM - 500 mMimidazole). Imidazole will compete with Histidine ring to bind toNi2+Low concentration of imidazole in the lysis and wash buffers removesnon-specific bound contaminant proteins, which may have some His-residues.A Nice Example of aSuccessful Purification*of an Enzyme Using aNi-Affinity Column*This gel photo appeared inActa Cryst.(2013)F69, 730 – 732Flow-through from load,washElutionloadwashelute5 mM30mMgradientImidazole34
10/29/20213Protein Concentration Determination: Bradford AssayBradford Assay(CoomassieBlue G-250)Lab 8: Purify AdhPprotein, determineits concentration595 nmAdhP Purification-Next StepsThis week:you performed Ni-NTAchromatography to purifyAdhP andchecked the presence of the proteinusingBradfordassay.Next week: you willcheck the purity of the protein on aprotein gel: look for thesize of the protein of interestandother contaminations coming from the preparationYou will confirm that you have the right protein using aWestern blot, an antibody will detect the presence ofthe V5epitope in AdhP protein56

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Term
Spring
Professor
ChristineM.Theodore
Tags
SDS PAGE

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