Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

1. Equilibrate the column with 5 column volumes of binding buffer. 2. Apply the sample. 3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 4. Elute with 5–10 column volumes of elution buffer. Collect fractions in neutralization buffer if low pH elution is used*. The purified fractions can be buffer exchanged using desalting columns (see page 134). *Since elution conditions are quite harsh, collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral. Since Benzamidine Sepharose 4 Fast Flow (high sub) has some ionic binding characteristics, the use of 0.5 M NaCl and pH elution between 7.4–8.0 is recommended. If lower salt concentrations are used, include a high salt wash step after sample application and before elution. The elution buffer used for competitive elution has a high absorbance at 280 nm. The eluted protein must be detected by other methods, such as an activity assay, total protein or SDS-PAGE analysis. The advantage with competitive elution is that the pH is kept constant throughout the purification. Cleaning Wash with 3–5 column volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 followed with 3–5 column volumes of 0.1 M sodium acetate, 0.5 M NaCl, pH 4.5 and re-equilibrate immediately with 3–5 column volumes of binding buffer. Remove severe contamination by washing with non-ionic detergent such as 0.1% Triton X-100 at +37 °C for 1 minute. Media characteristics Ligand density Composition pH stability* Mean particle size Benzamidine > 12 µmoles Amide coupling of ligand Short term 1–9 90 µm Sepharose 4 p-aminobenzamidine/ml via a 14 atom spacer to Long term 2–8 Fast Flow highly cross-linked 4% (high sub) agarose *Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Chemical stability All commonly used aqueous buffers. Storage Wash media and columns with 20% ethanol in 0.05 M sodium acetate, pH 4.0 (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. 56
Serine proteases and zymogens with an affinity for arginine Arginine Sepharose 4B Arginine Sepharose 4B is an L-arginine derivative of Sepharose 4B that can be used for any biomolecule with a biospecific or charge dependent affinity for arginine, such as serine proteases and zymogens. Specific examples include prekallikrein, clostripain, prothrombin, plasminogen and plasminogen activator. The L-arginine is coupled via its a-amino group, leaving the guanidino and a-carboxyl groups free to interact with samples. Electrostatic and stereospecific effects may contribute to the binding and elution process depending upon the specific sample involved. Figure 32 shows the partial structure of Arginine Sepharose 4B. S epharose 4 O CH2 CH CH2 O (CH 2) O OH CH 2 CHOH Fig. 32. Partial structure of Arginine Sepharose 4B. CH 2 NH CH _ COO NH CH2 CH2 CH2 NH C NH2 Purification option Binding capacity/ml Maximum operating Comments medium flow Arginine Sepharose 4B No data available 75 cm/h* Supplied as a suspension ready for column packing. *See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm. Performing a separation Determine the capacity of the medium for the sample of interest over a range of different pH and flow rates. The sample must be at the same pH as the binding buffer for each experiment. 1. Pack the column (see Appendix 3) and wash with 5 column volumes of binding buffer. 2. Equilibrate the column with 10 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A 280 nm ). 5. Elute with 10–20 column volumes of elution buffer. Biomolecules bound non-specifically can be eluted by: • step or gradient elution with increasing ionic strength (up to 1 M NaCl) • increasing concentration of urea or guanidine hydrochloride (up to 0.7 M) 57
Page 1 and 2:
Affinity Chromatography Principles
Page 3 and 4:
Affinity Chromatography Principles
Page 5 and 6:
Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
Page 14 and 15: BioProcess Media for large-scale pr
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 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
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 Column: rP
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47 and 48: Cleaning These procedures are appli
Page 49 and 50: Purification examples Figures 24 an
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54: Protein A fusion proteins IgG Sepha
Page 55 and 56: Purification or removal of serine p
Page 57: Sample: 2 ml clarified E. coli homo
Page 61 and 62: DNA binding proteins HiTrap Heparin
Page 63 and 64: Sample: Column: Binding buffer: Elu
Page 65 and 66: Media characteristics Ligand densit
Page 67 and 68: Sample: Pooled and frozen human pla
Page 69 and 70: The harsh conditions required to br
Page 71 and 72: Purification or removal of albumin
Page 73 and 74: IFN-act. A units 280 nm 250 0.12 20
Page 75 and 76: Purification options Binding capaci
Page 77 and 78: Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80: If detergent or denaturing agents h
Page 81 and 82: Glycoproteins or polysaccharides Co
Page 83 and 84: For complex samples containing glyc
Page 85 and 86: For complex samples containing glyc
Page 87 and 88: Chemical stability Avoid exposure t
Page 89 and 90: Proteins and peptides with exposed
Page 91 and 92: Several methods can be used to dete
Page 93 and 94: Media characteristics Composition M
Page 95 and 96: Performing a separation Binding buf
Page 97 and 98: Do not store the suspension for lon
Page 99 and 100: Sepharose has been modified and dev
Page 101 and 102: Ligand coupling Several methods are
Page 103 and 104: Couple a ligand through the least c
Page 105 and 106: Table 9 summarizes recommended liga
Page 107 and 108: Coupling through the primary amine
Page 109 and 110:
Ligand and column preparation 1. Di
Page 111 and 112:
Options Product Spacer Coupling con
Page 113 and 114:
100 80 Coupled substance, % 60 40 2
Page 115 and 116:
Coupling small ligands through amin
Page 117 and 118:
Ligand coupling 1. Add the ligand s
Page 119 and 120:
Alternative coupling solutions: Dis
Page 121 and 122:
Media characteristics Product Compo
Page 123 and 124:
Ligands containing amino groups can
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:
Appendix 1 Sample preparation Sampl
Page 133 and 134:
Remove salts from proteins with mol
Page 135 and 136:
1. Filter (0.45 µm) or centrifuge
Page 137 and 138:
For laboratory scale operations, Ta
Page 139 and 140:
Removal of lipoproteins Lipoprotein
Page 141 and 142:
Appendix 2 Selection of purificatio
Page 143 and 144:
4. Estimate the amount of slurry (r
Page 145 and 146:
Appendix 5 Conversion data: protein
Page 147 and 148:
Middle unit residue (-H 2 0) Charge
Page 149 and 150:
Expected results when changing cond
Page 151 and 152:
Expected results with competitive e
Page 153 and 154:
Appendix 8 Analytical assays during
Page 155 and 156:
Appendix 9 Storage of biological sa
Page 157 and 158:
Ordering information Product Quanti
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
show all

Affinity Chromatography - Department of Molecular and Cellular ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?