INSTRUCTIONS
`
`0678.1
`
`3747 N. Meridian Road
`P.O. Box 117
`Rockford, IL 61105
`
`UltraLink® Biosupport
`Medium
`53110
`53112
`53111
`Number
`Description
`UltraLink® Biosupport Medium
`53110
`Contents:
`UltraLink® Biosupport Medium, 1.25 g (approximately 10 ml of gel)
`Disposable Column Trial Pack, includes the following:
` Disposable Polystyrene Columns, 0.5-2.0 ml, 2 each
` Disposable Polypropylene Columns, 1.0-5.0 ml, 2 each
` Disposable Polypropylene Columns, 2.0-10.0 ml, 2 each
` Accessory Pack, 1 each
`UltraLink® Biosupport Medium
`Contents:
`UltraLink® Biosupport Medium, 6.25 g (approximately 50 ml of gel)
`Disposable Column Trial Pack, includes the following:
` Disposable Polystyrene Columns, 0.5-2.0 ml, 2 each
` Disposable Polypropylene Columns, 1.0-5.0 ml, 2 each
` Disposable Polypropylene Columns, 2.0-10.0 ml, 2 each
` Accessory Pack, 1 each
`UltraLink® Biosupport Medium, 0.25 g (approximately 2 ml of gel)
`Storage: Upon receipt products should be stored at room temperature. Products are shipped at
`ambient temperature. After opening, store the support at 4°C with desiccant. Rehydrated support
`should be stored at 4°C.
`Products are guaranteed for one year from the date of purchase when handled and stored properly.
`Introduction..................................................................................................................................................................................1
`Procedure Summary.....................................................................................................................................................................2
`Important Product Information ....................................................................................................................................................2
`Additional Materials Required.....................................................................................................................................................3
`General Coupling Instructions .....................................................................................................................................................3
`Related Pierce Products ...............................................................................................................................................................4
`Appendix .....................................................................................................................................................................................4
`Product References ....................................................................................................................................................................11
`General References....................................................................................................................................................................11
`Introduction
`Pierce UltraLink® Biosupport Medium is a preactivated, ready-to-use bead designed for use in affinity chromatography.
`These beads are composed of a bis-acrylamide/azlactone copolymer that is slightly hydrophobic in its active form and highly
`cross-linked. Because the azlactone functionality is copolymerized with the matrix material, the binding capacity is an
`integral part of the bead. This results in a high level of functionality throughout the porous bead matrix.
`
`53111
`
`53112
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
`www.piercenet.com • Customer Service: cs@piercenet.com • Technical Assistance: ta@piercenet.com
`1
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`Page 1 of 11
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`JSR Exhibit 1026
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`

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`The support is provided in dry form and reacts rapidly with nucleophiles, which allows coupling to a wide range of proteins
`and small molecules. The beads average 50-80 microns in diameter with a very open architecture that provides both high
`surface area and high pore volume. Due to the rigid polymeric nature of this support, it has excellent utility in medium-to-
`low pressure chromatography applications. These beads are further described in US Patent No. 4,871,824 and European
`Patent EP0392,735 B1.
`For most ligands, the addition of a chaotropic salt can greatly increase the coupling efficiency (Figure 7, Coupling Buffer
`Information, Appendix G). Other factors that can impact coupling efficiency are also described.
`Procedure Summary
`1. Add protein solution to dry beads and mix for 1 hour.
`2. Add quench solution and mix for 2.5 hours.
`3. Wash beads 5 times.
`4. Resuspend beads in buffer of choice.
`Important Product Information
`Chemistry
`The azlactone functionality of the UltraLink® Biosupport Medium couples nucleophiles on ligands via a ring opening
`reaction to attach the ligand to the support through stable covalent linkages. For example, amino-functional ligands will form
`stable amide bonds. Due to the unique azlactone chemistry, there is no leaving group or toxic chemical byproduct as a result
`of the coupling reaction, therefore this coupling chemistry is extremely safe and easy to use.
`
`Figure 1. Reaction of azlactone ring on UltraLink® Biosupport Medium with amine-containing ligand.
`Product Characteristics
`A. Stability
`•
`pH stability of matrix: 1-13
`• Temperature stability: 4-40°C
`• Reusability: Over 100 cycles with 99% capacity (Refer to Reusability Information, Appendix H for additional details)
`B. Coupling Capacities
`• Protein A: 240 mg/g beads = 30 mg/ml gel
`•
`IgG: 88 mg/g beads = 11 mg/ml gel
`• Bovine Serum Albumin (BSA): 120 mg/g beads = 15 mg/ml gel
`B. Specifications
`• Average Particle Size 50-80 microns
`• Functionality: >250 µmoles/g = >31.25 µmoles/ml gel = >0.25 mEq/g = >0.03 mEq/ml gel
`• Molecular Exclusion Limit: >2,000,000 daltons
`• Average Surface Area : >250 m2/g
`• Average Pore Volume: >1.2 cc/g (60% of bead volume)
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
`www.piercenet.com • Customer Service: cs@piercenet.com • Technical Assistance: ta@piercenet.com
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`Page 2 of 11
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`• Pore Size: 1,000 Å
`• Swell Volume: 8-10 ml gel/g beads*
`• Maximum Pressure: 100 psi (6.9 bar)
`• Maximum Linear Velocity: 3,000 cm/hour
`• Endotoxin Level (by LAL): <0.62 EU/ml gel
`*Check label for specific lot swell volume value
`
`Additional Materials Required
`• Amine-containing ligand to be coupled in appropriate buffer (see Coupling Buffer Information, Appendix G)
`• Vortex
`• Rocking platform or rotating device
`• Quench solution (3.0 M ethanolamine, pH 9.0)
`• Centrifuge, filtration device with < 25 micron filters or Handee™ Spin Cup Columns (Product No. 69700) for small
`volume samples
`• Wash solutions, such as phosphate buffered saline (PBS) [BupH™ Phosphate Buffered Saline Packs, 0.1 M phophate,
`0.15 M NaCl; pH 7.2, Product No. 28372] and 1.0 M sodium chloride (NaCl).
`
`General Coupling Instructions
`Note: For best results, please read this entire instruction booklet prior to using this product.
`Note: The entire coupling reaction, including all incubation times, can be completed within 6 hours.
`1. Weigh out sufficient beads for the column. (Refer to the individual lot swell volume value to determine the quantity of
`beads needed for the column.) Coupling can be accomplished in a centrifuge tube or beaker.
`2. Add protein solution directly to the dry beads. There is no need to swell the beads prior to use; the coupling buffer in the
`sample will swell the beads.
`Note: For most ligands, the addition of a chaotropic salt can greatly increase the coupling efficiency (See Figure 7,
`Coupling Buffer Information, Appendix G).
`3. Vortex sample at medium speed for a few seconds to suspend beads in the coupling solution. Gently rock or rotate for 1
`hour at RT. Coupling can also be done at 4°C or 37°C if ligand is susceptible at RT.
`Note: DO NOT USE MAGNETIC STIRBARS. They may damage the beads.
`4. Centrifuge sample at 1,200 x g at RT for 5-10 minutes or until beads are pelleted. Alternatively, filtration using a 25
`micron frit or, for small volumes, Handee™ Spin Cup Columns may be used to separate the protein solution from the
`beads.
`5. Remove supernatant by aspiration or decanting. Use care to retain beads in tube. This supernatant may be used for
`determination of the amount of ligand not coupled to the beads.
`Note: Due to the use of Triton® X-100 surfactant in the production of the beads, there may be interference with an A280
`measurement of the uncoupled protein. Therefore, the Pierce BCA Protein Assay (Product No. 23225) is recommended.
`6. Add quench solution to the beads at 10 times bead volume to block unreacted azlactone sites. Refer to Quenching and
`Quench Solutions, Appendix F, for additional details.
`7. Vortex and gently rock or rotate sample for 2.5 hours.
`8. Centrifuge sample at 1,200 x g for 5-10 minutes or until beads are pelleted. Remove and discard supernatant after
`centrifugation.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`Page 3 of 11
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`9. Resuspend beads in 10 times the bead volume of PBS.
`10. Vortex sample at medium speed for a few seconds to resuspend beads in the wash solution and gently rock or rotate
`sample for 15 minutes.
`11. Centrifuge samples as described in Step #8.
`12. Resuspend bead pellet in 1.0 M NaCl. The use of a high-salt wash solution, such as 1.0 M NaCl, will remove non-
`specifically attached protein.
`13. Vortex and gently rock or rotate sample for 15 minutes as in Step #10.
`14. Centrifuge samples as in Step #8.
`15. Resuspend bead pellet in PBS for 15 minutes as in Step #9.
`16. Vortex and gently rock or rotate sample for 15 minutes as in Step #10.
`17. Centrifuge beads as in Step #8.
`18. Repeat PBS wash as in Steps #15-17.
`19. Resuspend beads in final buffer of choice.
`
`Related Pierce Products
`28372
`BupH™ Phosphate Buffered Saline, 0.1 M phosphate, 1.5 M NaCl, pH 7.2
`23225
`BCA Protein Assay*
`69700
`Handee™ Spin Cup Columns
`ImmunoPure® IgG Elution Buffer, 1 L
`21004
`ImmunoPure® IgG Elution Buffer, 3.75 L
`21009
`29920
`Disposable Polystyrene Columns, 0.5-2.0 ml, 100/pkg
`29922
`Disposable Polypropylene Columns, 1.0-5.0 ml, 100/pkg
`29924
`Disposable Polypropylene Columns, 2.0-10.0 ml, 100/pkg
`29925
`Disposable Column Trial Pack, 2 columns each plus accessories
`
`Appendix
`A. Pressure-Flow Relationship
`UltraLink® Biosupport Medium is ideal for low- and medium- pressure chromatography. The following graph illustrates the
`relationship between the differential pressure on the beads and the linear flow velocity of fluid through the column.
`
`Figure 2. Pressure-flow relationship across UltraLink® Biosupport Medium.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`B. Effect of Time and Temperature on Protein Coupling
`Ligands couple rapidly and with high efficiency to UltraLink® Biosupport Medium at various temperatures including room
`temperature (RT), 4°C or 37°C. Recommended conditions for coupling are 1 hour at RT. These conditions may be adjusted
`as necessary for the specific ligand.
`
`Figure 3. Effect of time and temperature on protein coupling to UltraLink® Biosupport Medium.
`Experimental Conditions: Goat anti-rabbit antibody was coupled to UltraLink® beads using 10 mg beads + 1 mg protein/ml
`in 0.6 M sodium citrate, 0.1 M MOPS; pH 7.5 at 4°C ( ), RT ( )and 37°C ( ) for varying periods of time. Coupled ligand
`was radiometrically determined.
`C. Coupling of Proteins as a Function of pI and Molecular Weight
`The UltraLink® support azlactone-functional beads have the capacity to couple proteins of various isoelectric points (pI) and
`molecular weights effectively and efficiently.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`Page 5 of 11
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`Table 1. Coupling of proteins as a function of pI and molecular weight.
`Protein
`Molecular Weight
`Protein Offered
`(kD)
`(mg/ml)
`
`% Coupling
`Efficiency
`
`Capacity (mg/ml)
`
`Pepsin, pI 2.9
`
`Fetuin, pI 3.3
`
`BSA, pI 4.9
`
`Apoferritin, pI 5.0-
`5.5
`
`Human IgG, pI 5.8-
`7.3
`
`HRP, pI 6.1-7.2
`
`Carbonic anhydrase, pI
`6.2
`
`Myoglobin, pI 6.8-
`7.8
`
`Ribonuclease,
`pI 9.5
`
`34
`
`49
`
`66
`
`440
`
`160
`
`40
`
`29
`
`17
`
`14
`
`1.60
`
`0.76
`
`1.82
`
`1.47
`
`1.10
`
`1.73
`
`1.67
`
`1.81
`
`1.77
`
`46
`
`87
`
`68
`
`43
`
`92
`
`35
`
`75
`
`48
`
`57
`
`8.6
`
`7.9
`
`14.3
`
`7.4
`
`11.7
`
`6.8
`
`14.9
`
`9.9
`
`11.6
`
`14
`
`1.59
`
`56
`
`10.3
`
`Lysozyme,
`pI 11.0
`Experimental Conditions: The proteins were prepared in 0.8 M sodium citrate, 0.1 M bicarbonate; pH 8.6 as the coupling
`buffer, (due to solubility, the human IgG was prepared in 0.5 M sodium citrate). The General Coupling Instructions were
`followed with a 1 hour coupling reaction at RT using approximately 10.5 mg beads per sample. The amount of protein
`covalently coupled to the UltraLink® beads was determined by difference using the Pierce BCA Protein Assay Reagent
`(Product No. 23225).
`D. Effect of Protein Concentration
`Due to the high level of azlactone functionality, proteins couple with high coupling capacity and efficiency (80%) to
`UltraLink® Biosupport Medium over a wide range of concentrations. With IgG, maximal coupling capacity had not been
`reached at 11 mg IgG bound per ml gel or 88 mg IgG bound per gram of dry beads.
`
`Figure 4. Effect of protein concentration on coupling to UltraLink® Biosupport Medium.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`6
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`Page 6 of 11
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`Experimental Conditions: Goat anti-rabbit IgG was prepared in 0.6 M sodium citrate, 0.1 M MOPS; pH 7.5 as the coupling
`buffer at various protein concentrations. The protein solution was added to the dry UltraLink® beads for coupling following
`the General Coupling Instructions with a 1 hour coupling reaction at RT, 2.5 hours quenching with 3.0 M ethanolamine, pH
`9.0 and washing as per the recommended coupling procedure. To determine the amount of protein covalently coupled to the
`beads, protein-coupled beads were exposed to 1% sodium dodecyl sulfate (SDS) for 4 hours at 37°C. After incubation, beads
`were washed three times with 1% SDS for 15 minutes each wash. The amount of SDS resistant or covalently coupled protein
`was radiometrically determined.
`
`E. Effect of Volume of Coupling Protein Solution
`Even though the coupling buffer swells the beads, protein coupling can be accomplished efficiently in concentrated or dilute
`protein solutions and in varying volumes of coupling buffer. Use of very low or dilute protein solutions, i.e. 0.1 mg/ml may
`result in a slight decrease in coupling efficiency with a 1 hour coupling time.
`
`Figure 5. Effect of Volume of Coupling Solution on Efficiency of Coupling to UltraLink® Biosupport Medium
`Experimental Conditions: To 10 mg of UltraLink® beads, 200 µg protein (goat anti-rabbit IgG) was added in varying
`amounts of coupling buffer (0.6 M sodium citrate, 0.1 M MOPS; pH 7.5). Coupling conditions were as outlined in the
`General Coupling Instructions.
`F. Quenching and Quench Solutions
`Due to the high level of azlactone functionality in the beads, it is recommended to quench or inactivate excess azlactone
`groups after protein coupling. The beads can be quenched with 3.0 M ethanolamine, pH 9.0 at RT for a minimum of 2.5
`hours. In many cases, the time of quenching can be extended with little, if any, effect on protein coupling or subsequent use
`for affinity chromatography separations.
`Other quench solutions may be used including 1.0 M ethanolamine; pH 9.0, 0.1 M glucosamine in 1.0 M sodium sulfate; pH
`8.5, primary amine buffers such as 1.0 M Tris; pH 8.0 and alkylamines; e.g.; butylamine or methylamine.
`G. Coupling Buffer Information
`Several factors can affect coupling of the ligand to a support, especially the composition of the coupling buffer. Because of
`the unique coupling reaction with the azlactone functionality of the UltraLink® Biosupport Medium, the coupling buffer
`solution can be selected and tailored so that it is the most conducive for the ligand and for its coupling to this support. The
`choice of coupling buffer, including salt concentration and pH, can be easily adjusted without compromising the coupling
`reaction. Below are a few indicators of the versatility in selection of coupling buffers.
`Compatible Coupling Buffers
`Due to the chemical nature of the azlactone coupling reaction, a wide range of buffers may be used to covalently couple the
`ligand to UltralLink® Biosupport Medium. Some of the buffers that have been used to couple proteins are listed below.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`Table 2. Coupling buffers compatible with UltraLink® Biosupport Medium
`
`MOPS
`TAPS
`TES
`CHES
`POPSO
`Sodium carbonate
`Sodium acetate
`Sodium citrate
`Bicine
`Phosphate buffered saline
`
`CAPS
`HEPES
`TAPSO
`AMPSO
`MES
`Sodium pyrophosphate
`Sodium bicarbonate
`Sodium borate
`Tricine
`
`Important Considerations for Coupling Buffers:
`• Use of primary amine buffering systems, such as Tris, allows the primary amine of the buffer to compete with the protein
`for coupling to the azlactone functionality.
`• Sodium azide, phenols, potassium thiocyanate, ß-mercaptoethanol or ammonium sulfate should not be present in the
`coupling buffer as these chemicals may react with the azlactone functionality of the UltraLink® beads.
`• For most ligands, addition of a chaotropic salt will greatly increase the coupling efficiency (see Figure 7: Effects of Salts
`on Coupling). However, if your ligand is not compatible with high salt, the coupling can be done without the addition of
`extra salts.
`Effect of Coupling Buffer pH on Ligand Binding
`Proteins can be efficiently and effectively coupled to UltraLink® beads under a wide pH range. This allows the selection of a
`coupling buffer that is most compatible with the ligand. An example presented below demonstrates coupling of goat anti-
`rabbit IgG antibody to the azlactone-functional beads with coupling buffers at varying pH conditions. The most efficient
`coupling for this antibody was found to be at a pH of 7.5 or greater.
`
`Figure 6. Effect of coupling buffer pH on binding of ligands to UltraLink® Biosupport Medium.
`Experimental Conditions: The procedures outlined in the General Coupling Instructions were followed using goat anti-
`rabbit IgG at 1 mg/ml in coupling buffer and 10 mg UltraLink® beads. Coupling buffers with 0.6 M sodium citrate or 0.15 M
`NaCl include:
`
`pH 6.0 = 0.3 M MES
`
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`pH 7.0 = 0.1 M phosphate
`pH 7.5 = 0.1 M MOPS
`pH 8.0 = 0.1 M TES
`pH 8.5 = 0.2 M Tricine
`pH 10.0 = 0.2 M carbonate
`Effect of Salt Concentrations in the Coupling Buffer
`The addition of salts to the coupling buffer may enhance the amount of ligand covalently attached to the Pierce UltraLink®
`Biosupport Medium, regardless of the pH of the coupling buffer. Addition of salt to the coupling buffer should be based on
`salt compatibility of the ligand. Goat anti-rabbit IgG was coupled to the UltraLink® beads in three different buffers. To each
`of these buffers was added three different salts. The efficiency of covalent coupling increased with each buffer as a function
`of increasing salt concentrations as illustrated in the following graph.
`
`Figure 7. Effect of Salt Concentration in the Coupling Buffer for UltraLink® Biosupport Medium.
`
`H. Reusability Information
`UltraLink® Biosupport Medium is an excellent choice for affinity separations, especially when actual chromatography
`conditions are used and the immobilized ligand needs to be reused. To illustrate this point, rProtein A™ (recombinant Protein
`A) was coupled to UltraLink® beads and used to recover murine IgG from tissue culture fluid. The specific capacity eluted
`from this column after greater than 130 cycles remained at 99% of the original capacity.
`Table 3. Elution capacity of UltraLink® Biosupport Medium after multiple uses.
`Area of Eluted Peak (x10-6)
`Cycle Number
`IgG Eluted
`Capacity (mg/ml)
`1
`5.47
`1.43
`100
`20
`5.66
`1.45
`116
`50
`5.26
`1.42
`96
`100
`5.28
`1.43
`97
`139
`5.41
`1.37
`99
`Experimental Conditions: A 1.0 x 1.3 cm column was used with 1 ml of rProtein A™ coupled UltraLink® gel. The
`purification cycle was defined as: Load with 25 ml tissue culture fluid; ATCC HB-124 DB9G8 (murine anti-insulin IgG2ak)
`cultured in RPM1-1640 + 10% fetal calf serum (Gibco) using an Endotronics Acusyst-P5 hollow fiber reactor. The tissue
`culture fluid was filtered through a 0.45 micron Millipore Pelicon 6 cassette prior to application to the column. Wash the
`column with PBS; pH 7.2. Elute with 0.1 M sodium citrate; pH 2.5. Clean with 3.0 M guanidine HCI in 20 mM phosphate;
`
`% Initial Capacity
`
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`pH 7.2. Equilibrate with PBS; pH 7.2. The load phase of the cycle was run at 1 ml/minute (60 column volumes [CV]/hour)
`and all other phases were run at 3 ml/minute (180 CV hour).
`I. Resin/Column Maintenance Topics
`Column Preparation Procedures
`Columns can be prepared using either gravity or flow packing procedures. It is recommended that a 75% v/v slurry of beads
`be used to prepare columns fitted with a < 25 micron frit. When preparing a column for operation at higher flow rates (> 500
`cm/hour), it is recommended that the gel be packed at a flow rate 25% higher than the operational flow rate for a minimum of
`10 column volumes.
`Storage Conditions after Ligand Coupling
`Once the ligand has been coupled to the UltraLink® Biosupport Medium, storage of the ligand-coupled support at 4°C is
`recommended. If desired, a preservative solution may be included with storage at 4°C. Extended storage in the following
`solutions and subsequent binding in affinity separations has been documented.
`Table 4. Effect of storage conditions on binding capacity of immobilized ligand on UltraLink® Biosupport Medium.
`Storage Solution
`Specific Capacity
`Specific Capacity
`(in PBS)
`with Storage at 4°C
`with Storage at 4°C
`(mg/ml eluted IgG) at 1 month
`(mg/ml eluted IgG) at 3 months
`20% Ethanol
`18.5
`21.6
`0.05% Sodium Azide
`20.0
`19.1
`0.01% Benzyl Alcohol
`19.1
`20.9
`0.05% Thimerosal
`19.9
`20.2
`0.01 N Sodium Hydroxide
`19.8
`14.6
`Experimental Conditions: rProtein A™ was coupled to UltraLink® beads as per the General Coupling Instructions. Before
`storage, the specific capacity of the rProtein A™ coupled beads was determined using rabbit IgG and the beads allocated to
`the various storage conditions. 1 ml columns were poured and washed with 40 column volumes of PBS to remove the
`preservative solution before the 10 ml of 3 mg/ml rabbit IgG was loaded in 10 mM phosphate, pH 7.5. Specific capacities,
`using 0.1 M glycine, 2% acetic acid elution, pH 2.2, were determined by A280 measurements and chromatograms.
`Note: It is not recommended that 2% chlorhexidine gluconate in 20% ethanol be used as a preservative as precipitation of
`this preservative may occur with storage at 4°C.
`Cleaning Stability
`UltraLink® Biosupport Medium coupled to protein ligands can be successfully exposed to a wide variety of cleaning
`solutions without loss of specific eluted capacity. A stable protein (rProtein A™) was coupled to the beads, columns
`prepared, loaded with human IgG and specific capacities determined as outlined in the preceding Experimental Conditions.
`Each column was exposed to the following cycling conditions for 5 cycles:
`1. Equilibration with PBS, pH 7.5
`2.
`IgG loading in PBS, pH 7.5 (10 ml at 3 mg/ml)
`3. Elution with: 0.1 M glycine, 2% acetic acid, pH 2.2
`4. Cleaning solution for 10 column volumes
`5. Column equilibration with PBS, pH 7.5
`Generally, high salt concentration and acidic and basic cleaning solutions have no effect on column capacities. Chaotropes
`can be successfully used at appropriate concentrations.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
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`10
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`Table 5. Effect of cleaning solution on binding capacity of immobilized ligand on UltraLink Biosupport Medium.
`Cleaning Solution
`Specific Capacity
`Specific Capacity
`(mg IgG/ml gel)
`(mg IgG/ml gel)
`Cycle 1
`Cycle 5
`4.0 M NaCl in PBS
`19.9
`19.9
`4.0 M NaCl in 0.1 N HCI
`18.2
`18.9
`6.0 M Guanidine-HCI*
`19.3
`17.6
`2.0 M Sodium Thiocyanate
`18.2
`17.9
`8.0 M Urea
`18.5
`17.7
`20% Ethanol in PBS**
`19.2
`17.5
`0.1 N Sodium Hydroxide
`17.8
`18.4
`1.0 N Ammonium Hydroxide
`18.6
`20.2
`0.1 N o-Phosphoric Acid
`17.6
`19.1
`Refer to the Reusability section for additional information on the effect of column cleaning with guanidine-HCI.
` *
`Refer to Storage section for additional information on the use and effect of 20% ethanol in PBS.
`**
`Product References
`Ju tongzhong, et al. (2002). Purification, characterization, and subunit sturcture of rat core 1 β1,3-galactosyltransferase, J. Biol. Chem. 277(1), 169-177.
`Kornfeld, Rosalind, et al. (1998). Purification and multimeric structure of bovine N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase. J.
`Biol. Chem. 273(36), 23203-23210.
`General References
`Coleman, P.L., Walker, M.M., Milbrath, D.S. andStauffer, D.S. (1990). Immobilization of protein A at high density on azlactone-functional polymeric beads
`and their use in affinity chromatography. J. Chromatogr. 512, 345-363.
`Coleman, P.L., Walker, M.M., Heilmann, S.M., Krepski, L.R., Rasmussen, J.K. and Jensen, K.M. (1988). Affinity chromatography on a novel support:
`azlactone-acrylamide copolymer beads. FASEB J. 2, A1770 (#8563).
`Coleman, P.L., Milbrath, D.S., Walker, M.M., Heilmann, S.M., Rasmussen, J.K. and Krepski, L.R. (1990). Azlactone copolymer beads: applications in
`bioseparations. J. Cell. Biochem. 44, 19 (S14D).
`Coleman, P.L., Walker, M.M., Reese, C.L. and Milbrath, D.S. (1991). Effect of polyanionic salts on immobilization of protein A and antibody on
`azlactone-functional beads. FASEB J. 5, A805 (#2528).
`Hermanson, G.T., Mallia, A.K. and Smith, P.K. (1992). Immobilized affinity ligand techniques, Academic Press: San Diego, California, U.S.A., pp. 28-30,
`90-95,
`Milbrath, D.S., Coleman, P.L., Walker, M.M. and Stauffer, D.S. (1990). Azlactone-functional supports useful in affinity chromatography and other
`bioseparations. AIChE Extended Abstracts, #104E.
`Milbrath, D.S., Coleman, P.L., Walker, M.M., Heilmann, S.M., Rasmussen, J.K. and Krepski, L.R. (1989). Azlactone polymer supports for bioseparations.
`ACS Abstracts.
`Rasmussen, J.K., Heilmann, S.M., Krepski, L.R., Jensen, K.M., Mickelson, J. and Johnson, K. (1991/1992). Crosslinked, hydrophilic, azlactone-functional
`polymeric beads: A two-step approach. Reactive Polymers 16, 199-212.
`Rasmussen, J.K., Hembre, J.I., Koski, N.I. and Milbrath, D.S., et al. (1992). Mechanistic studies in reverse-phase suspension copolymerization of
`vinyldimethylazlactone methylenebis (acrylamide). Makromol. Chem., Macromol. Symp. 54/55, 535-550.
`Rasmussen, J.K., et al. (1990). Hydrophilic, crosslinked, azlactone-functional beads - A new reactive support. Polymer Reprints 31(2), 442-443.
`
`US Patent No. 4,871,824 .
`European Patent EP0 392,735 B1.
`
`Triton® is a registered trademark of Rohm & Haas.
`rProtein A™ is trademark of RepliGen Corp.
`BCA Technology is protected by U.S. Patent # 4,839,295.
`
`© Pierce Biotechnology, Inc., 10/2002. Printed in the USA.
`
`Telephone: 800-8-PIERCE (800-874-3723) or 815-968-0747 • Fax: 815-968-7316 or 800-842-5007
`www.piercenet.com • Customer Service: cs@piercenet.com • Technical Assistance: ta@piercenet.com
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