Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

Purification or removal of fibronectin Gelatin Sepharose 4B Fibronectin is a high molecular weight glycoprotein found on the surfaces of many cell types and present in many extracellular fluids including plasma. Fibronectin binds specifically to gelatin at or around physiological pH and ionic strength. Purification option Binding capacity/ml medium Maximum operating flow Comments Gelatin Sepharose 4B 1 mg human plasma fibronectin 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 Binding buffer: PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4 Elution buffer alternatives: - 0.05 M sodium acetate, 1.0 M sodium bromide (or potassium bromide), pH 5.0 - Binding buffer + 8 M urea - Binding buffer + arginine Fibronectin has a tendency to bind to glass. Use siliconized glass to prevent adsorption. Cleaning Wash 3 times with 2–3 column volumes of buffer, alternating between high pH (0.1 M Tris- HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5). Re-equilbrate immediately with 3–5 column volumes of binding buffer. Remove denatured proteins or lipids by washing the column with 0.1% Triton X-100 at +37 °C for one minute. Re-equilibrate immediately with 5 column volumes of binding buffer. Media characteristics Ligand density Composition pH stability* Mean particle size Gelatin 4.5–8 mg gelatin/ml Gelatin linked to Sepharose Short term 3–10 90 µm Sepharose 4B using the CNBr method Long term 3–10 *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 Stable in all commonly used aqueous buffers. Storage Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C. 68
Purification or removal of albumin HiTrap Blue HP, Blue Sepharose 6 Fast Flow The same procedure can be used either to purify albumin or to remove albumin as a specific contaminant before or after other purification steps. Albumin binds to Cibacron Blue F3G-A, a synthetic polycyclic dye that acts as an aromatic anionic ligand binding the albumin via electrostatic and/or hydrophobic interactions. Similar interactions are seen with coagulation factors, lipoproteins and interferon. Cibacron Blue F3G-A is linked to Sepharose to create Blue Sepharose affinity media. O NH 2 SO 3 Na SO 3 Na N NH O NH NH N SO 3 Na N O Sepharose Fig. 40. Partial structure of Blue Sepharose Fast Flow and Blue Sepharose High Performance. Use HiTrap Blue HP 1 ml or 5 ml columns to remove host albumin from mammalian expression systems, or when the sample is known to contain high levels of albumin that may mask the visualization of other protein peaks seen by UV absorption. Albumin can be a significant contaminant during the purification of immunoglobulins from ascites fluid, cell cultures or serum, chiefly because of its abundance in the original source material. Advice on the selection of techniques for the removal of albumin during antibody purification is given in The Antibody Purification Handbook from Amersham Pharmacia Biotech. Cibacron Blue F3G-A also shows certain structural similarities to naturally occurring molecules, such as the cofactor NAD + , that enable it to bind strongly and specifically to a wide range of proteins including kinases, dehydrogenases and most other enzymes requiring adenylyl-containing cofactors (see page 72). Purification options Binding capacity Maximum Comments operating flow HiTrap Blue HP Human serum albumin, 20 mg/column 4 ml/min (1 ml column) Prepacked columns. Human serum albumin, 100 mg/column 20 ml/min (5 ml column) Blue Sepharose 6 Human serum albumin, > 18 mg/ml medium 400 cm/h** Supplied as a Fast Flow* suspension ready for column packing. *A convenient alternative to Blue Sepharose CL-6B, since rehydration is not required. **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. 69
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 and 58: Sample: 2 ml clarified E. coli homo
Page 59 and 60: Serine proteases and zymogens with
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: The harsh conditions required to br
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?