Affinity Chromatography Handbook

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22 Troubleshooting This section focuses on practical problems that may occur when running a chromatography column. The diagrams below give an indication of how a chromatogram may deviate from the ideal during affinity purification and what measures can be taken to improve the results. Target elutes as a sharp peak. Satisfactory result A 280 If it is difficult or impossible to retain biological activity when achieving this result, either new elution conditions or a new ligand must be found. If using low pH for elution, collect the fractions in neutralization buffer (60–200 µl 1 M Tris-HCl, pH 9.0 per ml eluted fraction). Target is a broad, low peak that elutes while binding buffer is being applied A 280 Binding buffer Binding buffer Flow through (unbound material) Flow through (unbound material) Elution buffer Eluted target Eluted target Target elutes in a broad, low peak A 280 A 280 A 280 A 280 Binding buffer Binding buffer Binding buffer Binding buffer Flow through (unbound material) Flow through (unbound material) Flow through (unbound material) Flow through (unbound material) Elution buffer Elution buffer Elution buffer Eluted target Elution buffer Eluted target ml ml ml Wait Eluted target Eluted target ml ml ml Find better binding conditions. Try different elution conditions. If using competitive elution, increase the concentration of the competitor in the elution buffer. Stop flow intermittently during elution to allow time for the target molecule to elute and so collect the target protein in pulses (see second figure beneath). Note: This result may also be seen if the target protein has denatured and aggregated on the column or if there is non-specific binding. Some of the target molecule elutes as a broad, low peak while still under binding conditions Allow time for the sample to bind and/or apply sample in aliquots, stopping the flow for a few minutes between each sample application (see second figure beneath).
Situation Cause Remedy Protein does not bind Sample has not been filtered properly. Clean the column, filter the sample or elute as expected. and repeat. Sample has altered during storage. Prepare fresh samples. Sample has wrong pH or buffer Use a desalting column to transfer sample into conditions are incorrect. the correct buffer (see page 133). Solutions have wrong pH. Calibrate pH meter, prepare new solutions and try again. The column is not equilibrated sufficiently in the buffer. Repeat or prolong the equilibration step. Proteins or lipids have Clean and regenerate the column or use a precipitated on the column. new column. Column is overloaded with sample. Decrease the sample load. Microbial growth has occurred Microbial growth rarely occurs in columns in the column. during use, but, to prevent infection of packed columns, store in 20% ethanol when possible. Precipitation of protein in the Clean the column, exchange or clean the filter column filter and/ or at the top of the bed. or use a new column. Low recovery of activity, but Protein may be unstable or Determine the pH and salt stability of the normal recovery of protein. inactive in the elution buffer. protein. Collect fractions into neutralization buffer such as 1 M Tris-HCl, pH 9 (60–200 µl per fraction). Enzyme separated from Test by pooling aliquots from the fractions and co-factor or similar. repeating the assay. Lower yield than expected. Protein may have been Add protease inhibitors to the sample and degraded by proteases. buffers to prevent proteolytic digestion. Run sample through a medium such as Benzamidine 4 Fast Flow (high sub) to remove serine proteases. Adsorption to filter during sample preparation. Use another type of filter. Sample precipitates. May be caused by removal of salts or unsuitable buffer conditions. Hydrophobic proteins. Use chaotropic agents, polarity reducing agents Protein is still attached to ligand. or detergents. More activity is Different assay conditions have Use the same assay conditions for all the recovered than was been used before and after assays in the purification scheme. applied to the column. the chromatographic step. Removal of inhibitors during separation. Reduced or poor flow Presence of lipoproteins or Remove lipoproteins and aggregrates during through the column. protein aggregates. sample preparation (see Appendix 1). Protein precipitation in the column caused by removal of stabilizing agents during fractionation. Modify the eluent to maintain stability. Clogged column filter. Replace the filter or use a new column. Always filter samples and buffer before use. Clogged end-piece or adaptor or tubing. Remove and clean or use a new column. Precipitated proteins. Clean the column using recommended methods or use a new column. Bed compressed. Repack the column, if possible, or use a new column. Microbial growth. Microbial growth rarely occurs in columns during use, but, to prevent infection of packed columns, store in 20% ethanol when possible. 23
Page 1 and 2: 18-1022-29 Edition AD Affinity Chro
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: DNA binding proteins ..............
Page 7: Appendix 4 ........................
Page 10 and 11: 8 This handbook describes the role
Page 12 and 13: 10 Affinity chromatography is also
Page 14 and 15: 12 BioProcess Media for large-scale
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23: A 280 nm 0.6 0.4 0.2 0 Fig. 8. Scou
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 0.0 0 200
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: HiT
Page 43 and 44: Fig. 21. SDS-PAGE of non-reduced sa
Page 45 and 46: Purification options Binding capaci
Page 47 and 48: Performing a separation Binding buf
Page 49 and 50: Poly (His) fusion proteins His Micr
Page 51 and 52: One step, on-column, refolding and
Page 53 and 54: Purification using HisTrap Kit HisT
Page 55 and 56: 1. Pack the column (see Appendix 3)
Page 59 and 60: 1. Equilibrate the column with 5 co
Page 61 and 62: Specifically bound biomolecules can
Page 65 and 66: Sample: 2000 ml partially purified
Page 67 and 68: Coagulation factors HiTrap Heparin
Page 71 and 72: Purification or removal of fibronec
Page 73 and 74: Purification examples Figure 42 sho
Page 75 and 76:
Media characteristics Ligand and de
Page 77 and 78:
Media characteristics Ligand densit
Page 79 and 80:
Purification options Binding capaci
Page 81 and 82:
Elution buffers: use low concentrat
Page 83 and 84:
Page 85 and 86:
Lentil lectin for binding of branch
Page 87 and 88:
Page 89 and 90:
Remove proteases as quickly as poss
Page 91 and 92:
Purification example A 280 nm 1.0 0
Page 93 and 94:
Samples: 2.5 ml cell extract contai
Page 95 and 96:
In Activated Thiol-Sepharose 4B the
Page 97 and 98:
Reactivation Pass one to two column
Page 99 and 100:
Chapter 4 Components of an affinity
Page 101 and 102:
If several functional groups are av
Page 103 and 104:
Chapter 5 Designing affinity media
Page 105 and 106:
Coupling the ligand 1. Prepare the
Page 107 and 108:
Increase the ligand concentration t
Page 109 and 110:
Options Product Spacer arm Coupling
Page 111 and 112:
Media characteristics Product Ligan
Page 113 and 114:
Preparation of the matrix should be
Page 115 and 116:
Immunoaffinity chromatography Immun
Page 117 and 118:
Preparation of coupling reagent Use
Page 119 and 120:
Coupling through hydroxy, amino or
Page 121 and 122:
Coupled glycylleucine % Coupled lig
Page 123 and 124:
Coupling through a thiol group Thio
Page 125 and 126:
Chapter 6 Affinity chromatography a
Page 127 and 128:
Resolution is achieved by the selec
Page 129 and 130:
For any capture step, select the te
Page 131 and 132:
Sample clarification Centrifugation
Page 133 and 134:
Examples of precipitation agents ar
Page 135 and 136:
Resolubilization of protein precipi
Page 137 and 138:
Alternative 1: Manual desalting wit
Page 139 and 140:
Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
Page 143 and 144:
Appendix 5 Conversion data: protein
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Middle unit residue (-H 20) Charge
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Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
Page 153 and 154:
Appendix 9 Storage of biological sa
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Additional reading Code No. Purific
Page 157 and 158:
Product Quantity Code No. Blue Seph
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
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Affinity Chromatography Handbook

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