Presentation on theme: "Protein Purification Initial Questions How much and how pure?"— Presentation transcript:
1Protein Purification Initial Questions How much and how pure? applicationsourcefeasibilityNative configuration?functional/structural (yes)sequence (no)antibody (maybe)Detection method?functional assayband on gelWhy purify proteins?functional and/or structural studiesindustrial or pharma-ceutical applicationsgenerate antibodiespartial sequence
2Developing a Protein Purification Scheme carry out small pilot experiments to evaluate various separation techniquesstart with rapid high capacity techniques (which are generally low resolution) and progress to high resolution low capacity techniques
4Developing a Protein Purification Scheme carry out small pilot experiments to evaluate various separation techniquesstart with rapid high capacity techniques and progress to high resolution low capacity techniquesminimize time and number of manipulations whenever possibleeg, arrange methods to minimize buffer changes if other factors are equalexploit unique features
5Exploiting Unique Features affinity chromatographyCaM Ca2+/hydrophobicsubunits vs. complex (gel filtration)
6Evaluation of Protein Purification qualitative (gel electrophoresis)quantitativerecovery (% yield)fold-purification% yield =total act. recoveredtotal starting act.fold-purification =sp. act. recoveredstarting sp. act.
7Protein Sequencing partial sequence data: identify protein by homology design DNA probesassess purityautomated Edman degradationprotein bound to solid supportN-terminal residues sequentially removedidentified by HPLCpossible after gel electrophoresis
8Preparative Electrophoresis high resolution provides analytical informationdifficult to exploit in protein purificationcapacityrecoveryrecovery of proteins from gelsdiffusionelectroelutionimmunizationtransfer to membrane
9Protein Transfer electrophoretic transfer using special apparatus PVDF membranesgood protein retentionchemical resistancetransfer in buffer with 10% MeOH to reduce SDSoptimize transfer timesmaller proteins transfer fasterlarger proteins retained better0.22 and 0.45 mm pore sizeproduces replica of gel
10N-terminal Sequencing Needs relatively pure sample (>80%)pmoles of protein0.5-5 mg for 50 kDa‘unblocked’ N-terminusfree of contaminants (Tris, glycine, SDS, acrylamide, etc.)
11Microsequencing Procedure Purify protein so that it is a major band resolved from contaminants.Gel electrophoresis (1- or 2-D)Transfer protein to membrane support following electrophoresis.Stain membrane and excise protein band of interest.Wash membrane extensively with H2O before sequence analysis.Submit membrane to sequencing service.
12Internal Sequencingtreat protein with site-specific protease or chemicals
13Internal Sequencingpeptides generally isolated by reverse phase (HPLC) chromatographypeaks dried onto glass fiber filters and sequenced