GE Healthcare Life Sciences Data file 18-1163-8 AC Gel filtration Superose High-performance Columns Superose prepacked columns are designed for highperformance, laboratory-scale separations of proteins and other biomolecules according to size. Due to its wide separation range, Superose is a versatile medium. Superose prepacked columns give fast and reproducible separations with good resolution. The columns are ideal for the final polishing step in a purification procedure, where the remaining trace contaminants are removed. Superose columns offer: High-resolution separations of proteins and other biomolecules according to size Wide separation range separations in two molecular weight ranges from 1 up to 5 million M r Excellent reproducibility and durability Laboratory scale purifications, from µg to mg Media characteristics Superose prepacked columns are available with two different media: Superose 6 or Superose 12. Table 1 summarizes the main characteristics of Superose. Good resolution separations of proteins and other biomolecules according to size Gel filtration separates biomolecules according to molecular size. The method is ideal for the final polishing step in a purification procedure when sample volumes have been reduced. Since the composition of the buffer does not Fig 1. Superose 6 GL and Superose 12 GL prepacked Tricorn columns. directly affect the separation, elution conditions such as ph and salt concentration can be varied to suit the sample or the requirements for the next step. A high-performance medium like Superose is invaluable when it is necessary to obtain protein preparations free of dimers and other aggregates, for example before crystallization trials. Superose 6 and Superose 12 have an average bead size of 13 µm and 11 µm respectively, and are designed for laboratory-scale separations. The small bead size gives good resolving capacity. Superose 6 prep grade and Superose 12 prep grade have an average bead size of 3 µm and are suitable for larger scale separations.
Table 1. The main characteristics of Superose media Superose 6 Superose 12 Separation range (M r ) 5 to 5 (globular proteins) 1 to 3 (globular proteins) Exclusion limit (M r ) 4 (globular proteins) 2 (globular proteins) Chemical stability Stable in all common buffers: 1 M acetic acid, 8 M urea, 6 M guanidine hydrochloride, 1% SDS, organic solvents,.5 M NaOH (for cleaning-in-place) Stable in all common buffers: 1 M acetic acid, 8 M urea, 6 M guanidine hydrochloride, 1% SDS, organic solvents,.5 M NaOH (for cleaning-in-place) ph stability 3 to 12 (long-term), 1 to 14 (short-term) 3 to 12 (long-term), 1 to 14 (short-term) Temperature stability working storage 4 C to 4 C 4 C to 3 C 4 C to 4 C 4 C to 3 C Matrix Cross-linked agarose Cross-linked agarose Average particle size 13 µm 11 µm Wide separation range separation in two ranges from M r 1 up to M r 5 million Superose 6 and Superose 12 provide good resolution, high selectivity gel filtration separations over a very broad molecular weight range (Superose 6: 5 5 and Superose 12: 1 3 ), as shown in Figure 2. Superose complements Superdex by offering wider molecular weight separation ranges. A comparison between Superose and Superdex is shown in Figure 2. Superose 6 is especially valuable for its ability to separate very large biomolecules according to size. Superdex Peptide Superdex 75 Superdex 2 Superdex 3 prep grade Superdex 75 prep grade Superdex 2 prep grade Superose 6 Superose 12 Superose 6 prep grade Superose 12 prep grade 1 3 1 4 1 5 1 6 1 7 Fig 2. Separation ranges (globular proteins) of Superose and Superdex gel filtration media. High chemical and ph stabilities Superose is formed from cross-linked agarose matrices that have high chemical and broad ph stabilities (short-term ph 1 to 14, long-term ph 3 to 12). Superose media can be used with all common aqueous buffers and are unaffected by detergents such as 1% SDS, chaotropic salts or dissociating agents such as 8 M urea or 6 M guanidine hydrochloride, and organic solvents such as 3% acetonitrile. The media can withstand strong bases (such as.2 M NaOH) and strong acids (such as 1 M acetic acid or 7% formic acid) allowing the use of stringent cleaning procedures, which are essential to maintain column efficiency without reducing the lifetime of the column. Column characteristics Superose media are prepacked in high performance glass columns. Both column types Tricorn (GL) and Precision (PC) are made of glass, which allows visual inspection of the gel bed. All parts of the columns are biocompatible. Table 2 shows the characteristics of Tricorn and Precision columns prepacked with Superose. Tricorn columns are simple to use, with fittings for simple connection to ÄKTAdesign and other high-performance LC systems, and can be run according to their specifications where the systems have the appropriate pressure capacity. Precision Columns were designed for SMART systems, but the Precision Column Holder allows use of the columns with a variety of high-performance systems. All parts of the columns are biocompatible. 2 18-1163-8 AC
Table 2. Characteristics of Tricorn and Precision columns prepacked with Superose Superose 6 1/3 GL columns Tricorn Columns Superose 12 1/3 GL columns Superose 6 PC 3.2/3 columns Precision Columns Superose 12 PC 3.2/3 columns Bed dimensions (i.d. height mm) 1 3 1 3 3.2 3 3.2 3 Bed volume (ml) 24 24 2.4 2.4 Recommended sample volume (μl)* 25 5 25 5 2 5 2 5 Theoretical Plates (m -1 ) > 3 > 4 > 3 > 4 Recommended flow rate (ml/min)*.1.5.5 1..1.1.1.1 Max. flow rate (ml/min)** 1 1.5.1.1 Max. pressure over column 15 bar 3 bar 12 bar 24 bar (217 psi, 1.5 MPa) (435 psi, 3 MPa) (174 psi, 1.2 MPa) (348 psi, 2.4 MPa) * In general, a lower sample volume gives a higher resolution. ** (H 2 O at 25 C). Excellent reproducibility and durability Reproducible results are essential in all research. The long working life and high reproducibility of Superose prepacked columns, both run-to-run and column-to-column, are a result of optimized column design, the stable nature of Superose, and well-proven, reliable column packing procedures. These are in addition to the controlled column manufacturing and media synthesis, and strict quality control testing of every production batch of Superose and each prepacked column. Figure 3 shows the separation of protein standards on Superose 12 1/3 GL before and after 21 consecutive runs. Operation Choice of eluents Eluents can be chosen freely to improve recovery from separations of crude sample or to overcome solubility problems. For example, chaotropic agents and detergents can be used to improve the solubility of membrane proteins..15 M NaCl, or a buffer with equivalent ionic strength, is recommended to avoid ph-dependent non-ionic interactions with the matrix. Sample volumes The quality of a gel filtration separation is largely independent of sample concentration, but to achieve good resolution, the sample volume should be less than 5% of the total column volume. Sample volumes between.1% and 1.% of the bed volume (25 to 25 µl) give the highest resolution in Superose 1/3 GL columns. For more information see Table 2. Column: Sample: Sample volume: 5 µl A 28 mau 15 1 5 A 28 mau 15 1 5 Superose 12 1/3 GL 1. IgG (M r 15 ) 2.5 mg/ml, 2. BSA (M r 67 ) 8 mg/ml, 3. β-lactoglobulin (M r 35 ) 2.5 mg/ml, 4. Cytochrome C (M r 12 7) 1 mg/ml, 5. Vitamin B 12 (M r 1355).3 mg/ml Eluent:.15 M NaCl +.5 M phosphate ph 7. Flow rate: System: Detection:.5 ml/min ÄKTAFPLC A 28 nm Test separation, run 1 1 2 3 4. 5. 1. 15. 2. ml Test separation, run 21 1 2 3 4. 5. 1. 15. 2. ml Fig 3. Comparison of the test separation runs 1 and 21 after repeated injection of a test sample on Superose 12 1/3 GL. No difference in chromatographic performance is observed after 21 runs. 5 5 18-1163-8 AC 3
Large-scale preparative separation To handle larger sample volumes it may be necessary to pack larger columns. Superose 6 prep grade and Superose 12 prep grade are available in lab packs for use when larger column volumes are required. Superose prep grade media have an increased bead size to give a similar selectivity to prepacked Superose columns, but easier column packing and tolerance to crude samples. Figure 4 shows selectivity curves for Superose prep grade (3 µm), compared to Superose 6 (13 µm) and Superose 12 (1 µm). K av 1..8.6.4.2 Superose 12 Superose 6 prep grade Superose 6 Superose 12 prep grade 1 3 1 4 1 5 1 6 Mr Fig 4. Selectivity curves for Superose 6 and 12, and Superose 6 and 12 prep grade (globular proteins). Applications Separation of standard proteins Superose 6 1/3 GL and Superose 12 1/3 GL are prepacked columns produced under optimum conditions to give the highest column efficiency and performance. These columns offer good resolution and broad selectivity gel filtration over a broad molecular weight range, as shown in Figure 5. A Column: Superose 6 1/3 GL Sample: Proteins from Gel Filtration Calibration Kits LMW and HMW (GE Healthcare) T = thyroglobulin (M r 669 ) F = ferritin (M r 44 ) Ald = aldolase (M r 158 ) O = ovalbumin (M r 43 ) CA = carbonic anhydrase (M r 29 ) R = RNase A (M r 13 7) Apr = aprotinin (M r 6 5) Sample volume: 1 µl Eluent:.5 M Phosphate,.15 M NaCl, ph 7.2 Flow rate:.5 ml/min System: ÄKTAexplorer 1 Detection: 28 nm 7 6 5 4 3 2 1 5 1 15 2 25 ml 7 6 5 4 3 2 1 mau B mau F Ald T O CA 5 1 15 2 25 ml F O Ald R Apr CA R Apr 1..9.8.7.6.5.4.3.2.1. 1 1 1 Log M r 1..9.8.7.6.5.4.3.2.1 K av K av Aprotinin RNase A Aprotinin RNase A Carb. anh Column: Superose 12 1/3 GL Sample: Proteins from Gel Filtration Calibration Kits LMW and HMW (GE Healthcare) F = ferritin (M r 44 ) Ald = aldolase (M r 158 ) O = ovalbumin (M r 43 ) CA = carbonic anhydrase (M r 29 ) R = RNase A (M r 13 7) Apr = aprotinin (M r 6 5)) Sample volume: 1 µl Eluent:.5 M Phosphate,.15 M NaCl, ph 7.2 Flow rate:.5 ml/min System: ÄKTAexplorer 1 Detection: 28 nm Ovalb Carb. anh Ovalb Aldolase Aldolase Ferritin Thyrogl Ferritin. 1 1 1 Log M r Fig 5. (A) Standard proteins separated on Superose 6 1/3 GL, molecular weight range: 5 5 and calibration curve. (B) Standard proteins separated on Superose 12 1/3 GL, molecular weight range: 1 3 and calibration curve. 4 18-1163-8 AC
Micropurification of human tears Figure 6 shows an example of the extremely small amounts of sample that can be analyzed on a Superose PC column. A 28nm A A 254 nm.5 434 458 54 54 587 Column: 2 Superose 6 high performance columns 1 3 mm (i.d. bed height) Sample: 1 µg Hae III, cleared pbr 322 Eluent:.5 M Tris-HCI, 1 mm EDTA, ph 8 Flow:.1 ml/min.25.2.15 267 192 184 234 213.1.5 123 124 2 4 6 Time (min) Fig 6. Micropurification of.75 µl of human tears using Superose 12 PC3.2/3 column on a SMART system. Separation of DNA fragments Figure 7 shows examples of separations performed using a Superose prepacked high-performance column for the separation of DNA fragments. In Figure 7a, two columns have been linked in series to increase resolution. 14 89 8 64 57 51 21 18 11 7 2 3 4 5 Time (h) B A 254 nm.5 1353 178 872 63 Column: Superose 6 high performance column 1 3 mm (i.d. bed height) Sample: φx-174 Rf DNA, Hae III digested, 1 µg Eluent:.5 M Tris, ph 8 Flow:.4 ml/min 31 281 271 234 194 118 72 3 6 Time (min) Fig 7. Two examples of separation of DNA fragments on a Superose high-performance column, 1 3 mm (inner diameter bed height). Peak figures correspond to number of base pairs. 18-1163-8 AC 5
Ordering information Product Quantity Code No. Superose 6 1/3 GL 1 17-5172-1 Superose 6 PC 3.2/3 1 17-673-1 Superose 12 1/3 GL 1 17-5173-1 Superose 12 PC 3.2/3 1 17-674-1 Related products Gel Filtration Calibration kit HMW 1 28-438-42 Gel Filtration Calibration kit LMW 1 28-438-41 Precision Column Holder 1 17-1455-1 Literature Tricorn Empty High Performance Columns, Data file 18-1147-36 Superdex High-performance columns, Data file 18-1163-79 Gel Filtration Handbook 18-122-18 Strategies for Protein Purification Handbook 28-9833-31 Recombinant Protein Purification Handbook 18-1142-75 Prepacked chromatography columns for ÄKTA design systems, Selection guide 28-9317-78 Gel Filtration Selection Guide 18-1124-19 For local office contact information, visit www.gelifesciences.com/contact www.gelifesciences.com/protein-purification GE Healthcare Bio-Sciences AB Björkgatan 3 751 84 Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. ÄKTAdesign, ÄKTAexplorer, ÄKTAFPLC, Superose, Superdex, and Tricorn are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners. The Tricorn column and components are protected by US design patents USD5856, USD56261, USD5555, USD4956 and their equivalents in other countries. 22 211 General Electric Company All rights reserved. First published Nov. 22 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5 D-79111 Freiburg Germany GE Healthcare Bio-Sciences Corp. 8 Centennial Avenue, P.O. Box 1327 Piscataway, NJ 8855-1327 USA GE Healthcare Japan Corporation Sanken Bldg., 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-73 Japan 18-1163-8 AC 3/211