ÄKTAdesign Purification - Harvard University

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 .. 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 UNICORN.

2 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 .. 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 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 ..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 .. Adjustment of the standard Purification protocol.

4 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 ..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 .

5 858. Introduction ..858.. The Purification development scheme .. Standard Purification protocol for synthetic phosphorothioate Purification protocol development scheme .. 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 .. 05 Optimization of individual protein Purification steps .. 08 Purification of peptides .. 4 Synthetic peptides .. 4 Protein fragments.

6 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 .. 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.

7 Basis of chromatography Basis of Ion Exchange Chromatography (IEC) .. Basis of hydrophobic chromatography (HIC).. Basis of reversed phase chromatography (RPC) .. Basis of size exclusion chromatography (SEC).. 46 Appendix III Glossary .. About this handbookThis handbook is intended to help you make full use of your KTAexplorer or KTApurifier chromatography system. Rather than being a text book in separation science, the handbook is instead a collection of useful step-by-step protocols to aid your everyday Purification work. Detailed instructions and recommendations are presented in a straightforward format , which should be easy to follow without needing a high level of expertise in programming or in specially adapted for the chromatography software UNICORN help you to plan, optimize and run your Purification Purification protocols are based on pre-constructed templates available in UNICORN.

8 In combination with the UNICORN Column list , these templates make method creation very handbook covers Purification of proteins, peptides and oligonucleotides. However, before developing a suitable Purification protocol, a development platform must be established, including: Required quality and quantity of the purified target molecule. Available information on the physical properties of the target molecule. Stability range for the starting material as well as the target molecule. Properties of the starting development of a suitable Purification protocol starts with running Standard Purification Protocols. These protocols take advantage of the high overall resolution obtained by combining techniques with independent selectivities. This will provide sufficient purity in a large number of cases. When satisfactory results are not obtained with the Standard Purification Protocols, different optimizing measures are you are not familiar with how to operate UNICORN, read Making your first run before using the protocols of this full description of how to operate UNICORN is found in the UNICORN User Manual.

9 Introduction to this handbook and UNICORN KTAdesign Purification User Manual 8- 24-23 Edition ADAbout this handbook5A brief theoretical background to the techniques used in this handbook is given in Appendix II on page 33 and further help is found in Appendix I on page About UNICORN templates for method Pre-constructed method templatesUNICORN contains a collection of pre-constructed method templates for different chromatographic situations. These templates are built up of blocks that are different parts used to build up a method, Column equilibration, Sample introduction, Fractionation, Elution, etc. Each method template contains a complete set of instruction variables within the blocks and, when an appropriate column is chosen from the Column list of UNICORN, column-specific default values (pressure limit , flow rate, etc.) are entered automatically. The default values represent the average chromatographic experiment and are used in many of the Standard Purification Protocols in this handbook.

10 Creating a method can be achieved in a few simple steps: . Select a chromatographic technique2. Select a suitable Select an appropriate column from the Column list .4. If necessary, change the variable Save the method under a new name. KTAdesign Purification User Manual 8- 24-23 Edition AD Introduction to this handbook and UNICORN .2 About UNICORN templates for method editing6 The pre-constructed templates are quite complex, so we do not recommend you to make any changes other than those allowed in the Run Set-up Variables page. However, you can create your own templates as described in the UNICORN User Method templates available in UNICORNUNICORN provides templates for the following chromatographic techniques: 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 are provided to support the use of different alternatives for sample injection and fraction collection.