GE SP HP Manual — Free PDF Download

Page 2 - Designation; Connectorkit; Connectors supplied; Table of contents; Selection of ion exchanger and conditions

p. 2 Code No. Designation No. supplied 17-1151-01 HiTrap SP HP 5 x 1 ml 17-1152-01 HiTrap SP HP 5 x 5 ml 17-1153-01 HiTrap Q HP 5 x 1 ml 17-1154-01 HiTrap Q HP 5 x 5 ml Connectorkit Connectors supplied Usage No. supplied 1/16” male/luer female Connection of syringe to top of HiTrap column 1 Tubing c...

Page 3 - Media properties; SP Sepharose; Column; The column cannot be opened or refilled.

p. 3 1. Description Media properties SP Sepharose ™ High Per formance and Q Sepharose High Per formance are strong cation and strong anion exchangers respectively. Both remain charged and maintain high capacity over broad pH ranges. The functional groups are coupled to the matrix via chemically stab...

Page 4 - HiTrap SP HP and HiTrap Q HP columns characteristics; pH stability, long term

p. 4 Table 1. HiTrap SP HP and HiTrap Q HP columns characteristics Column volumes 1 ml or 5 ml Column dimensions 0.7 × 2.5 cm (1 ml) and 1.6 × 2.5 cm (5 ml) Total ionic capacity 0.14–0.20 mmol (Cl – )/ml medium (Q) 0.15–0.20 mmol (H + )/ml medium (SP) Dynamic binding capacity SP: approx. 55 mg ribon...

Page 5 - Selection of buff er pH and ionic strength; The net charge or a protein as a function of pH.

p. 5 2. Selection of ion exchangerand conditions Ion exchange chromatography is based on adsorption and reversible binding of charged sample molecules to oppositely charged groups attached to an insoluble matrix. The pH value at which a biomolecule carries no net charge is called the isoelectric poi...

Page 6 - Strategy 1. Binding and elution of all sample components; Star t conditions are subject to the stability of the sample; Strategy 2. Enrichment of target protein

p. 6 Strategy 1. Binding and elution of all sample components Binding is achieved by choosing a star t buf fer with a low pH for HiTrap SP HP or a high pH for HiTrap Q HP. The ionic strenght should be kept as low as possible to allow all components to bind to the ionic exchange (<5 mS/cm). This r...

Page 7 - Starting pH; At least 1 pH unit below the pI of substance to be; pH interval

p. 7 The buf fering ion should carry the same charge as the ion exchange group and should have a pKa within 0.5 pH units of the pH used in the separation. Buf fering ions of opposite charge may take par t in the ion exchange process and cause local disturbances in pH. Starting pH Cation exchangers (...

Page 8 - Buf fers for anion exchange chromatography.; pH

p. 8 Table 3. Buf fers for anion exchange chromatography. pH interval Substance Conc. (mM ) Counter-ion pKa (25 °C ) 1 4.3–5.3 N-Methylpiperazine 20 Cl - 4.75 4.8–5.8 Piperazine 20 Cl - or HCOO - 5.33 5.5–6.5 L-Histidine 20 Cl - 6.04 6.0–7.0 bis-Tris 20 Cl - 6.48 6.2–7.2; 8.6–9.6 bis-Tris propane 20...

Page 10 - le

p. 10 Ta b le 4 . P re p a c ke d c o lu m n s f o r d e sa lt in g a n d b u ff e r e x c h a n g e . C o d e N o C o lumn Lo a d ing vo lume Elu tio n vo lume C o mme n ts A p p lic a tio n 1 7 -1 4 0 8 -0 1 H iT ra p 0 .1 –1 .5 m l 1 .3 – 4 .0 m l P re p a c ke d w it h F o r d e sa lt in g a n d...

Page 11 - Purifi cation

p. 11 Puriﬁ cation 1. Fill the syringe or pump tubing with star t buf fer (low ionic strength). Remove the stopper and connect the column to the syringe (with the provided adaptor), “drop to drop” to avoid introducting air into the column. 2. Remove the snap-of f end at the column outlet. 3. Wash ou...

Page 13 - Further optimization; Choice of gradient type; Another, but less common, method to desorb bound material is to

p. 13 Further optimization The recommendations given above will give a sound basis for developing an ef ficient purification step. Details of how flow rate, sample loading, par ticle size and elution scheme may be optimized to meet the special needs can be found in the handbook, Ion Exchange Chromat...

Page 14 - Elution with stepwise ionic strength gradients; Apply the sample at 1 or 5 ml/min for the HiTrap 1 ml or 5 ml column; Elution with continuous ionic strength gradients

p. 14 Elution with stepwise ionic strength gradients Stepwise elution is the sequential use of the same buf fer at dif ferent ionic strengths. It is technically simple and fast, and is suitable for syringe operation. It is of ten used for sample concentration and sample clean-up. Stepwise elution gi...

Page 15 - Determination of binding capacity; ELISA or other appropriate techniques.

p. 15 – A peristaltic pump and a gradient mixer e.g. pump P-1, gradient mixer GM-1. – A one pump system, e.g. ÄKTAprime ™ plus. – A two pump system, e.g FPLC or ÄKTA .1. Choose star ting conditions as outlined under Optimizing star ting conditions. 2. Equilibrate the column, see Purification.3. Adju...

Page 17 - Ordering Information; Product; HiTrap SP HP

p. 17 7. Scaling up For quick scale-up of purifications (back pressure will increase), two or three HiTrap ion exchange columns of the same type can be connected in series. For fur ther scale-up SP Sepharose High Per formance and Q Sepharose High Per formance are available in prepacked HiLoad ™ colu...

Page 18 - Related products; Accessories

p. 18 Related products No. Supplied Code No. HiTrap IEX Selection Kit 7 x 1 ml 17-6002-33 HiTrap Desalting 5 x 5 ml 17-1408-01 HiTrap Desalting 100 x 5 ml* 11-0003-29 HiPrep 26/10 Desalting 1 x 53 ml 17-5087-01 HiPrep 26/10 Desalting 4 x 53 ml 17-5087-02 PD-10 Desalting column 30 17-0851-01 HiLoad 1...

Page 19 - Realated literature

p. 19 Realated literature Ion Exchange Chromatography & ChromatofocusingHandbook, Principles and Methods 1 11-0004-21 Ion Exchange Columns and Media,Selection Guide 1 18-1127-31 Convenient Protein Purification,HiTrap Column Guide 1 18-1129-81