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ÄKTAdesign Purification - Harvard University

KTA KTAdesign PurificationMethod handbookGE HealthcareProtocol FinderPlanning protein purificationStandard Purification protocol for proteins .. 28 Adjustment and optimization .. 33 Planning peptide Purification synthetic peptidesStandard Purification protocol for synthetic peptides .. 47 Adjustment and optimization .. 54 Peptides from natural sourcesStandard Purification protocols for natural peptides .. 63 Adjustment and optimization .. 69 Protein fragmentsStandard Purification protocols for protein fragments .. 77 Adjustment and optimization .. 80 Planning oligonucleotide purificationStandard Purification protocol for synthetic phosphorothioate oligonucleotides.

• Anion exchange chromatography (AIEX) • Cation exchange chromatography (CIEX) • Hydrophobic interaction chromatography (HIC) • Reversed phase chromatography (RPC) • Size exclusion chromatography (SEC) • Affinity chromatography (AC) Besides a basic template for each chromatographic technique, templates

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1 KTA KTAdesign PurificationMethod handbookGE HealthcareProtocol FinderPlanning protein purificationStandard Purification protocol for proteins .. 28 Adjustment and optimization .. 33 Planning peptide Purification synthetic peptidesStandard Purification protocol for synthetic peptides .. 47 Adjustment and optimization .. 54 Peptides from natural sourcesStandard Purification protocols for natural peptides .. 63 Adjustment and optimization .. 69 Protein fragmentsStandard Purification protocols for protein fragments .. 77 Adjustment and optimization .. 80 Planning oligonucleotide purificationStandard Purification protocol for synthetic phosphorothioate oligonucleotides.

2 87 Anion exchange chromatography protocol .. 94 RPc protocol .. 95 Adjustment and optimization .. 96 Changing the scale .. 99 Trouble-shooting .. 101 Column maintenance .. 103 The MapNaturalSourcePureProteinPurePeptideFr agmentsQuantitativeAnalysisAmino AcidAnalysissPeptideMappingPeptideLibrar yPeptideSynthesisSequencingScreeningModi ficationsOptimization CycleActiveAnaloguesClonedGeneDNAS ynthesisSequencingProbeAntisensePrimersI solatedGeneDiagnosticSequencingor PCRA nalyticalDNA electro -phoresisGeneFishingNaturalSourceProtein /PeptideStructureGeneStructurePage 8 Gene fishingloopPage 61 Page 77 Page 77 Page 43 Page 21 Page 87 Page 87 Page 87 Page 87 KTAdesign Purification User Manual 8- 24-23 Edition ADContents1 Introduction to this handbook and

3 UNICORN ..5 . About this handbook ..5 .2 About UNICORN templates for method editing ..6 .2. Pre-constructed method templates ..6 . Method templates available in UNICORN ..7 .3 Abbreviations in UNICORN method .3. Abbreviations for General chromatography Templates ..8 . Abbreviations for special feature templates ..9 .4 Method creation ..9 .4. Example ..9 . Example 2 .. 0 .5 About UNICORN column list .. 2 .6 About automatic buffer preparation .. 3 .7 About adviser in UNICORN .. 42 The Purification of biomolecules ..152. Purpose of Purification .. The map .. How to develop Purification protocols.

4 Introduction .. General construction of Purification protocols .. Basic principles for the development of a Purification protocol ..2 3 Planning protein Purification ..233. Introduction ..233.. The Purification protocol development scheme ..233..2 General Purification protocol development scheme ..253..3 Planning platform protocol .. Standard Purification protocol for proteins .. Adjustment and optimization .. Adjustment of the standard Purification protocol .. Optimizing the final purity ..334 Planning peptide Purification ..414. Introduction ..4 4.. The Purification protocol development scheme.

5 4 KTAdesign Purification User Manual 8- 24-23 Edition ADContents25 Planning synthetic peptide Purification ..435. Introduction ..435.. Purification strategy ..43 General Purification protocol development scheme ..445..3 Planning platform protocol .. Standard Purification protocol for synthetic peptides .. Protocol I, for peptides soluble in neutral and acidic solvents .. Protocol II, for peptides soluble in neutral and alkaline solvents .. Addition alternative I, cation exchange ..5 Addition alternative II, size exclusion chromatography .. Adjustment and optimization.

6 Adjustment of the standard Purification protocol .. Optimizing the final purity .. Selectivity optimization .. Further optimization ..556 Planning Purification of peptides from natural sources ..596. Introduction ..596.. Purification strategy ..596..2 Purification development scheme ..606..3 Planning platform protocol .. Standard Purification protocol for natural peptides .. Adjustment and optimization .. Adjustment of the standard Purification protocol .. Optimizing the final purity .. Selectivity optimization .. Further optimization ..707 Planning Purification of protein fragments.

7 757. Introduction ..757.. Purification strategy ..757..2 Purification development scheme .. Purification protocol for protein fragments .. Adjustment and optimization .. Adjustment of the standard Purification protocol .. Optimizing the final purity .. Selectivity optimization ..8 Further optimization ..8 5..2 KTAdesign Purification User Manual 8- 24-23 Edition ADContents38 Planning synthetic oligonucleotide Purification ..858. Introduction ..858.. The Purification development scheme .. Standard Purification protocol for synthetic phosphorothioate Purification protocol development scheme.

8 Planning platform protocol .. Standard Purification protocol, synthetic phosphorothioate oligonucleotides .. Standard Purification protocol for synthetic oligonucleotides ..9 Purification protocol development scheme .. Planning platform protocol .. Anion exchange Purification protocol for synthetic oligonucleotides .. RPC Purification protocol for synthetic oligonucleotides .. Optimizing the final purity .. Selectivity optimization .. Further optimization ..969 Changing the scale ..9910 Trouble-shooting ..10111 Column maintenance ..10312 Applications ..105 2.. Purification of proteins.

9 05 Optimization of individual protein Purification steps .. 08 Purification of peptides .. 4 Synthetic peptides .. 4 Protein fragments .. Example of optimization of individual peptide Purification steps .. 2 Purification of synthetic oligonucleotides .. 22 Phosphorothioate oligonucleotides .. 22 Labelled oligonucleotides .. 23 KTAdesign Purification User Manual 8- 24-23 Edition ADContents4 Appendix I Tips and hints ..125I. Sample stability .. Conditioning the sample for a particular Purification step .. Sample pre-treatment and storage .. Requirements on the starting material.

10 Sample storage conditions .. Optimizing recovery .. Means of evaluating Purification results .. Techniques for the determination of total protein content .. Techniques for the determination of target peptide content .. 3 Techniques for the determination of complexity .. 3 Appendix II A brief theoretical background to the chromatography techniques ..133II. Properties of target molecules used for separation .. Sources of proteins and peptides .. Basis of chromatography Basis of Ion Exchange chromatography (IEC) .. Basis of hydrophobic chromatography (HIC).

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