Vivawell 8-strip IEX centrifugal multiwell purification strips For in vitro use only

Transcription

1 Technical data and operating instructions Vivawell 8-strip IEX centrifugal multiwell purification strips For in vitro use only

2 Vivawell 8-strip - Introduction The Vivawell 8-strip Principle Vivawell 8-strip IEX multiwell plates use the basic principle of ion exchange: separation accomplished on the basis of charges carried by solvent molecules. This is achieved using a centrifuge with a swing-out multiwell plate rotor equipped to take standard footprint deep-well plates. Membrane alternatives The Vivawell 8-strip IEX is available in three different membrane functionalities to cover every ion exchange application. (See table 1 on the next page for membrane chemistries). Vivawell 8-strip IEX plates are designed to eliminate the tedious chromatographic steps normally associated with ion exchange chromatography. In addition, the Vivawell 8-strip IEX product range is truly scaleable and is supported by individual Vivapure IEX spin columns, and fully validated large-scale membrane-based chromatography systems. Vivawell 8-strip IEX plates are ideal tools for developing methodologies to screen target proteins against different loading/ eluting conditions and membrane types. 8-Strip design features Vivawell 8-strip centrifugal plates feature a modular design. The plates are built up from 8-well units, strips, allowing the number of wells to be matched to the number of samples being processed. Vivawell 8-strips make multiwell plate technology far more economical when fewer than 96 samples need to be processed simultaneously. When working with less than 96 samples using traditional plates, either the partially used plates are disposed of, increasing the cost per preparation, or partially used plates must be treated for storage use and reuse, which raises problems of storage conditions and cross contamination of samples. The modular design of Vivawell 8-strip plates eliminates these problems. Used strips can be economically disposed of, while unused strips can be safely stored for future use. Equipment supplied x Vivawell 8-strips x 12 strip holding frames x 500 μl 96-well deep-well plates for loading, washing, and elution of samples. 4. Technical data and operating instructions. Additional equipment required 1. Centrifuge with swing-out rotor accepting stacks of 4 standard or 2 deep-well 96-well plates per carrier, and capable of spinning at 1,000 x g. 2. Multi-channel pipette or set of pipettes for dispensing small volumes of liquid ( μl; 200-1,000 μl) μm 96-well filter plates or syringe filters for sample clarification (Prod No , or equivalent). 2

3 Operation Functional groups Sulphonic acid (S) R-CH 2 -SO - 3 Na + Strong acidic cation exchanger Quaternary ammonium (Q) R-CH 2 -N + -(CH 3 ) 3 Cl - Strong basic anion exchanger Diethylamine (D) R-CH 2 -N-(C 2 H 5 ) 2 Weak basic anion exchanger 1. Position Vivawell 8-strip IEX Plate on top of a deep-well collection plate. Strips that are not required can be removed by pushing upwards from the bottom, taking care not to damage the drip nozzles on the underside of the strips. Unused strips should be stored at room temperature. Check for correct alignment of reference markings on both plates. 2. Equilibrate each of the wells to be used, by filling with 200 μl of loading buffer (See table 2 for buffer recommendations). 3. Centrifuge the stacked plates at 1,000 x g for 2 minutes and discard the flow-through. A:1 A:1 1 Note Centrifugation at higher speed is not recommended. Centrifugation at a lower speed will necessiate longer spin times, but will not alter the purification characteristics of the Vivawell plate Prepare samples in loading buffer either by diluting 1:6 in loading buffer, or buffer exchange using ultrafilters (Prod No. VS0101), or dialysis. Note Prepared samples should ideally have an ionic strength below 50 mm. Higher salt levels may restrict binding

4 Operation 5. Clarify the samples by filtration through 0.45 μm 96-well filter plates following the manufacturer s recommendation or 0.45 μm syringe filters (Product No or equivalent); this prevents blocking of the membrane pores and increases binding capacity. 6. Load 100 μl μl of prepared sample per well and centrifuge at 1,000 x g for 2 minutes. If the species of interest does not bind to the membrane under the conditions selected, it will pass through into the collection plate. This fraction should be retained. If the species of interest is bound to the membrane, empty and wash the collection plate, or exchange it for a fresh plate The remaining unbound fraction should be washed from the membrane with 2 x 200 μl volumes of fresh loading buffer centrifuging as before. Discard the wash fractions if not required. 8. Elute the bound protein fraction with μl aliquots of elution buffer per well centrifuging as before. (See table 2 for buffer recommendations). 6,7,8 Buffers and membranes Buffer recommendations Ion exchanger Select a buffer ph that is: Loading conditions Salt elution scheme S cation exchanger Below the iso-electric point Buffer in the ph range 3-8; Step-wise salt gradient elution of the binding species salt concentration ideally <25 mm to a final 1 M salt concentration * Q & D anion exchangers Above the iso-electric point Buffer in the ph range 5-10; Step-wise salt gradient elution of the binding species salt concentration ideally <25 mm to a final 1 M salt concentration * * In some cases up to 2 M salt may be required Ion exchange membranes Our ion exchange membranes consist of stabilised regenerated cellulose membranes to which charged groups have been covalently bound. The charged groups are associated with mobile counter-ions. The counter-ions may be reversibly exchanged with other ions of the same net charge. The charged protein of interest must displace the counter-ion and so bind to the exchanger on the membrane. Non-binding solutes can be washed from the membrane. Ionic or ph conditions are then altered so that the bound proteins are displaced and eluted. Elution is usually performed by a step-wise sequential increase in the salt concentration or by adjusting the ph of the buffer. 4 Choosing the correct ion exchanger for your application You would normally select a D or Q type exchanger if your protein has a low isoelectric point or a S type exchanger if your protein has a high iso-electric point. The iso-electric point is the ph at which the protein has no net charge in solution. Note It is also the ph at which the protein is least soluble and most easily precipitated. If you buffer a protein to below its isoelectric point, the protein will be positively charged and will bind to a S type exchanger. At a ph above the iso-electric point, the target protein will be negatively charged and will bind to a Q or D type exchanger. Proteins may bind to one or more of the ion exchange types in the Vivawell 8-strip IEX product range. If you have previously used a particular ion exchange resin with your target protein, we would recommend you to use the equivalent ion exchange membrane type. For example, if you are currently using a preparative DEAE-based resin, we advise you to select the D-type membrane. Please note that as proteins have diverse primary and tertiary structures, very few proteins behave identically during ion exchange using membranes and beaded media. To obtain the best purification through ion exchange, it is important your protein is stable over an extended ph range and in conditions of low ionic strength. Furthermore, the iso-electric point of your protein should be far from that of contaminants.

5 Buffers and membranes Technical specifications Vivawell 8-strip IEX Maximum sample volume per well Dynamic binding capacity per well Minimum recommended elution volume per well Storage conditions Dimensions Length Width Height Total height of Vivawell 8-strip IEX plus collection plate Materials of construction Vivawell 8-strip IEX plate Membrane supporting matrix Deep-well collection plate Holding frame Plate capacities 300 μl 1 mg 100 μl Room temperature 128 mm 85.5 mm 22 mm (+ 7 mm drip nozzle) 47 mm Polypropylene Stabilised regenerated cellulose Polypropylene Polystyrene Buffer recommendations We recommend you to use a buffering ion with the same charge as the membrane and with a pka within 0.5 ph units of the working ph. During elution of your target protein, the buffer concentration should maintain a constant ph when an increasing salt concentration is added. Generally, buffer concentrations between mm provide good buffering capacity in the ph range pka ± 1. Buffers with positive charges (e.g. amine buffers such as Tris) should not be used with S type exchanger. Negatively charged buffers (e.g. phosphate buffers) should not be used with Q or D type exchangers. Standard PBS buffer should not be used as it contains, along with other salts, 150 mm NaCl, which will significantly reduce binding to the ion exchange membrane. Vivawell devices are compatible with all commonly used aqueous buffer systems. There is no need to degas any buffers before use with Vivawell devices. Sample preparation You may need to dilute your sample up to 10 fold in order to lower its ionic strength to a level that will allow binding to the membrane. It is best to dilute your sample in loading buffer to a salt concentration equal to or less than 30 mm salt. We recommend you clarify your sample before loading. You should use a 0.45 μm 96-well filter plate following the manufacturer s recommendations, or use individual 0.45 μm syringe filters (Minisart Product No ). Alternatively, you can centrifuge your samples at 5,000 x g for 5 minutes to sediment any cellular debris or large visible particles, though this option may result in longer spin times for sample loading. 5

6 Chemical compatibility Table 4: Chemical compatibility Agent Concentration S Q D Alcohols Methanol 98 % Methanol 60 % Ethanol 99 % n-propanol 100 % n-propanol 60 % Iso-propanol 100 % Iso-propanol 60 % Butan-2-ol 99 % Glycerol 100 % Ethylene glycol 20 % Polyethylene glycol 20 % Ketones Acetone 100 % Methyl ethyl ketone 100 % Acids Acetic acid 1.0 M Formic acid 25 % Hydrochloric acid 1.0 M Sulphuric acid 1.0 M Trifluoroacetic 2.0 M Bases Ammonium hydroxide 28 % Ammonium hydroxide 10 % Sodium hydroxide 1.0 M Detergents n-octyl-β-d-glucopyronoside 2.0 % SDS 2.0 % Triton X % Tween % Oxidising agents Hydrogen peroxide 0.9 M Sodium hypochlorite 200 ppm Culture medium DMEM Norm RPMI-1640 Norm = YES, = NO (two hours at room temperature) 6

7 Ordering information Vivawell 8-strip IEX Description Pack size VW08IQ02 Vivawell 8-strip Q-IEX centrifugal purification strips 24 VW08IS02 Vivawell 8-strip S-IEX centrifugal purification strips 24 VW08ID02 Vivawell 8-strip D-IEX centrifugal purification strips 24 Vivawell-96 IEX VW96IQ02 Vivawell-96 Q-IEX 96-well protein purification plate 2 VW96IS02 Vivawell-96 S-IEX 96-well protein purification plate 2 VW96ID02 Vivawell-96 D-IEX 96-well protein purification plate 2 Vivawell vacuum manifold and vacuum compatible 8-strip plates Vivawell Vac 96/8-well system VW96VAC01 Vivawell 96/8-well vacuum manifold 1 VW96VAC02 Vivawell 96/8-well liquid trap and reservoir 1 VW96VAC03 96 deep well collection plate 1 ml (square wells) 25 VW96VAC04 96 deep well collection plate 2 ml (square wells) 25 VW96VAC05 Replacement seal for Vivawell Vac96/8-well Vacuum manifold 25 Vivawell Vac 8-strip system VW08VAC01 Vivawell Vac 8-strip vacuum manifold 1 VW08VAC02 Vivawell Vac 8-strip liquid trap and reservoir 1 VW08VAC03 8 well collection strips 1.2 ml (round wells) 125 VW08VAC04 Replacement seal for Vivawell 8-strip Vacuum manifold 1 Vivawell 8-strip IEX VW08IQ02 Vivawell 8-strip Q-IEX centrifugal purification strips 24 VW08IS02 Vivawell 8-strip S-IEX centrifugal purification strips 24 7

8 Sartorius Stedim Biotech GmbH August-Spindler-Strasse Goettingen, Germany Phone Fax Copyright by Sartorius Stedim Biotech GmbH, Goettingen, Germany. All rights reserved. No part of this publication may be reprinted or translated in any form or by any means without the prior written permission of Sartorius Stedim Biotech GmbH. The status of the information, specifications and illustrations in this manual is indicated by the date given below. Sartorius Stedim Biotech GmbH reserves the right to make changes to the technology, features, specifications and design of the equipment without notice. Status: July 2008, Sartorius Stedim Biotech GmbH, Goettingen, Germany Specifications subject to change without notice. Printed in Germany on paper that has been bleached without any use of chlorine. W G Publication No.: SL-5503-e08073 Order No.: