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Understanding Protein Purification and Sequencing Techniques

School
University of Wisconsin, Milwaukee**We aren't endorsed by this school
Course
CHEM 501
Subject
Biology
Date
Dec 10, 2024
Pages
12
Uploaded by HighnessLorisMaster72
Chapter5->Proteins:10structure->Istructure:aminoandsequenceofaproteinI↓proteinproteinpurificationsequencing①proteinpurification&a)changeoftheproteinofinterest"charged["-"charged.=>ion-exchangechromatography
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Ion Exchange Chromatography~->bird" - "chargedproteins"anion-exchangechromatography"org·-bird"+"chargedproteinsproteinofinterest.action-exchangeg>·proteinofinterestwillbirdo·tocolumn"chargedproteawillflow-through·eluteproteinofinterestwhincreasingconcofLionst
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Ion Exchange Chromatography
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Hydrophobic Interaction Chromatography-b)hydrophobicity-aprotein->polarproteinswillflow⑳->hydrophobicproteinswillbindathecolumn->hydrophobicproteinsareelutedw/increasingconcentrationofdetergent
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Size-exclusion Chromatography1)Telargeproteinporesizelargewillpassquicklythroughsmalopenthecolumn->smallprotein (poresizeretainonthecolumnorwithintheresinlongertime&elute
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Affinity Chromatographyd)AffinityDNAaffinitytagstation#I/IproteinofinterestitNterCterrI-His-His-His-His-His-His........-⑭Nizt·->elutewhincreasingconeofimidazole
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②proteinsequencing:toknowtheorderofAASinapolypeptidechaina)EdmandegradationRI#&H..basicOH-PITC↓S>EanationEDITO+in↓mem↑-PTH-aid
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SynthesizePE -aminoacidsfor20aminoacidad->"standards"->loadandHPLCorLC/MSIntensity-AlaPTH-Hispilt-tyrwil-HR1=-Hissidechain->Limitations:·hastobeverypureprotein·hasrestrictionstoshortpeptideb)MassSpectrometry
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·protein↓proteasedigestione.g.Trysic:cleavestermtoLysandArgA--D-E-F-kfL-M-N-RtS-T↓--D~~BIntensiv↓ms1M/Z↓frag mentationa
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⊥↓MmizAGCEDEE3774bionsy- ionsbiAC-D-E-F-y5b2A-CD-E-FKY4bsA-C-DE-F-k4334A-C-D-EF-KY235A-c-D-E-FkY
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Electrospray Ionization (ESI)Mass Spectrum
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Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved.Tandem Mass Spectrometry
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