™
®
Description
• Blue Trisacryl M is an affinity chromatography resin for the purification of a wide variety of proteins such as kinases (and other enzymes), albumin, interferons, and some coagulation factors.
• Blue Trisacryl M resin can be used for albumin depletion in situations in which albumin interferes with downstream applications, e.g., proteomics and protein purifications.
• The bead is composed of Trisacryl GF2000, a macroporous non-ionic support on which Cibacron
N
Blue has been covalently immobilized.
• Versatile use:
– Fully automated in combination with an automated chromatography instrument such as the ÄKTAdesign N systems.
– Semi-automated in combination with pumps.
– Manual operation in combination with a syringe.
Ordering Information
Part Number
25896-C001
Description Color
Blue Trisacryl M Dark Blue
Column Volume
1 mL
Pkg
5/pkg

Table of Contents
Section
Specifications
Technical Overview
Influence of Flow Rate and Residence Time on
Dymanic Binding Capacity of HSA and BSA
Working Conditions
Instructions for Use – Automated or Pumped
Chromatography Systems
Instructions for Use – Manual Use with Syringe
Procedure for the Determination of Target Protein Dynamic Binding
Capacity (DBC) Using an Automated Chromatography System
Regeneration and Maintenance
Sanitization
Storage Recommendations
Adapter Recommendations
Warning
Page
1 1
1 2
1 3
1 4
1 5
1 6
1 7
1 8
1 9
1 9
10
11
Note: The procedures herein are intended only as a guide. Users should always verify product performance with their specific applications under actual use conditions. If you have questions about the information presented in this guide, please contact Pall Life Sciences technical service.

Specifications
Materials of Construction
Column Housing, Cap, Plug, and Adapter: Polypropylene
Column Frit: Polyethylene
Blue Trisacryl M Resin Properties
Particle Size: 40-80 µm
Exclusion Limit: 10 7 Da
Ligand: Cibacron Blue
Capacity for Human Albumin: > 10 mg/mL † pH Stability: 1-10
Pressure Stability: Up to 3 bar (44 psi)
Stability to Detergents and Denaturing Agents: Excellent
Column Geometry
Column Volume (CV): 1.04 mL
Bed Height: 1.48 cm (0.58 in.)
Bed Diameter: 0.94 cm (0.37 in.)
Device Dimensions
Diameter: 1.6 cm (0.6 in.)
Length (Without Plugs): 4.8 cm (1.9 in.)
Connections
Inlet: Threaded female luer
Outlet: Rotating male luer locking hub
Flow Rates
Recommended flow rate for protein capture – 0.2-1.0 mL/min;
(residence time = 5.2-1.0 min)
Maximum Column Pressure
3 bar (300 kPa, 43.5 psi)
Storage
2-8 ºC (36-46 ºF); do not freeze
† Capacity determined using HSA @ 5 mg/mL in PBS. Column dimensions – 1.6 cm ID x 3 cm bed height; residence time (Tr) = 7.26 min.
1

Technical Overview
Blue Trisacryl M Resin is a mixed mode media. The ligand has some structural similarity with nucleotide co-factors such that several proteins, many of them enzymes, have a specific affinity for this ligand. The ligand structure includes hydrophobic and ionic moieties (see Figure 1) enabling less specific interactions with proteins like albumin. Common applications include protein purification and albumin depletion.
Figure 1
Structure of Cibacron Blue Ligand Used for Blue Trisacryl M Resin
The binding capacity of human albumin to Blue Trisacryl M Resin is higher than bovine albumin. Residence time (Tr) influences the efficiency of binding, as seen by the increase in capacity with longer residence times. Thus greater capacity can be achieved with lower flow rates. Additionally, multiple columns can be connected for higher capacity, particularly for depletion applications.
2

Influence of Flow Rate and Residence Time on Dynamic Binding Capacity
(DBC) of HSA and BSA †
HSA
BSA
Flow Rate
(mL/min)
0.5
1.0
2.0
4.0
0.5
1.0
2.0
4.0
Tr (min)
2.08
1.04
0.52
0.26
2.08
1.04
0.52
0.26
DBC @ 10% BT
5.4 +/- 0.3
4.5 +/- 0.3
3.5 +/- 0.3
2.9 +/- 0.0
6.3 +/- 0.0
4.6 +/- 0.3
3.5 +/- 0.3
2.6 +/- 0.3
DBC @ 50% BT
21.0 +/- 1.0
15.2 +/- 1.0
10.1 +/- 0.8
1 6.7 +/- 0.5
12.0 +/- 0.0
11.1 +/- 0.8
1 8.5 +/- 0.7
1 6.1 +/- 0.6
† Binding of 5 mg/mL albumin in PBS at indicated flow rate using 1 mL AcroSep
Blue Trisacryl M column. Three columns tested once at each speed. DBC are average of triplicates +/-1 standard deviation. Tr = (CSA x ht)/flow rate;
Tr = residence time, CSA = cross sectional area, ht = bed height
3

Working Conditions
Column Equilibration and Sample Application
– Equilibrate the column with 5 CVs of loading buffer. PBS is a frequent buffer choice.
– Inject the sample into the column.
Working Flow Rate
Blue Trisacryl M is a semi-rigid resin. It will withstand high flow rate without shrinkage. However, capacity is sensitive to residence time. Lower flow rate
(0.2-0.5 mL/min) for the sample loading step is recommended to achieve higher dynamic binding capacity.
Elution
Elution is generally achieved with high salt concentrations (e.g., 3 M NaCl).
Upon first use of Blue Trisacryl M Resin for a particular sample, the following procedure is recommended:
– Elute with sodium chloride up to 3 M, pH ~7.
– If necessary, optimize the elution conditions, particularly the final NaCl concentration.
– For proteins that interact more specifically with the ligand, competitive elution can also be performed using appropriate co-factors (e.g., NAD, NADP). More energetic eluents, such as ethylene-glycol, urea, and potassium isothiocyanate may be used as alternatives.
4

Instructions for Use – Automated or Pumped Chromatography Systems
Materials Required
• System (ÄKTAdesign, pump, or equivalent)
• Filtered, degassed buffers
Automated System Protocol
1. Attach column to pre-primed system. To prevent air from getting into the column, fill the neck of the column dropwise while system is running very slowly.
Allow the buffer to flow through the column until all bubbles in the bottom of the column have been evacuated.
2. Equilibrate with 5-10 CV of PBS or chosen buffer.
3. Load desired amount of sample at 0.2-0.5 mL/min. Higher capacities are observed with lower flow rates.
4. Wash column with 5-10 CV PBS or chosen buffer.
5. Elute with 10-20 CV chosen elution buffer, stepwise or gradient.
6. Strip with 5-10 CV of 3 M NaCl in buffer used above.
7. Re-equilibrate with 5-10 CV PBS
5

Instructions for Use – Manual Use with Syringe
Materials Required
• Syringes (5-30 mL) with luer lock fittings
• Filtered buffers
Syringe Protocol
Note: It is important to avoid introducing air into the column. Remove air bubbles from fluid filled syringe before attachment to the column each time the syringe is changed.
When pushing fluid through the syringe, maintain a relatively constant flow rate with minimal backpressure, typically 0.2-1.0 mL/min.
1. Fill a syringe with loading buffer.
2. Equilibrate the column with 5-10 CV of loading buffer.
3. Fill a syringe with sample.
4. Load sample onto the column at 0.2-0.5 mL/min, avoiding the introduction of air bubbles. Lower flow rates result in more efficient protein capture.
– Collect all effluent to assess possible target protein breakthrough if capacity is exceeded.
5. Wash column with 5 CV of loading buffer to remove unbound proteins.
– Collect all effluent for analysis.
6. Fill a syringe with elution buffer. Secure this syringe to the column luer connector. Check that there are no air bubbles at the site of attachment.
7. Run 10-20 CV of elution buffer through the column to elute bound protein.
– Collect all effluent containing eluted protein(s) in appropriated sized fractions.
8. If the column will be reused, strip residual protein with 10 CV of high salt buffer
(e.g., 3 M NaCl)
9. Fill the syringe with loading buffer. Equilibrate the column with 5-10 CV of loading buffer.
6

Procedure for the Determination of Target Protein Dynamic Binding
Capacity (DBC) Using an Automated Chromatography System
For most accurate prediction of DBC during a purification scheme, chose conditions that closely match conditions to be used during target protein purification.
System Parameters
• Protein loading step
– Flow rate: 0.2-0.5 mL/min (or intended flow rate)
– Equilibrate: 10 CV loading buffer
– Sample load: Inject sufficient quantity of protein-containing sample to exceed column capacity
– Wash: 10 CV loading buffer
– Elution: 10 CV elution buffer
– Re-equilibration: 10 CV loading buffer
• Void volume (V
0
): To determine V
0
, either perform a run in the by-pass position
(by-passes the column) or run the procedure using conditions which prevent protein binding. For example, use an elution buffer instead of a loading buffer for the equilibration and sample loading steps.
Calculation of AcroSep Column DBC
• Formula: DBC = C x (V
L
-V
0
)
C = Concentration of load
V
L
= Volume at 10% or 50% breakthrough
V
0
= From the void volume determination described above, V
0 is the total volume passing through the system from the time of injection (0% deflection of
OD
280
) until protein breakthrough (increase in OD
280
).
Note: To convert the column DBC to resin DBC, divide the DBC value above by the column volume (1.04 mL resin).
7

Regeneration and Maintenance
In order to avoid frequent regeneration and/or column fouling, use only samples and buffers which are filtered through a 0.2 µm membrane. Verify that the changes in pH and ionic strength which occur during the purification procedure will not cause precipitation of any components in the sample.
If necessary, after repeated uses Blue Trisacryl M resin may be regenerated. The regeneration procedure must be adapted to the type of adsorbed material to be eliminated. The following suggestions must be checked first for their degree of efficiency:
Situation
General Cleaning-In-Place (CIP)
Adsorbed Contaminants
Recommendation
Wash with 3 M sodium chloride and demineralized water. Make repeated and alternated washings with buffer solutions at acidic pH (2-3) and basic pH (9-10).
Wash with chaotropic agents such as
6-8 M urea, 6 M guanidine hydrochloride or
50% ethylene-glycol (v/v).
After regeneration treatment, re-equilibrate the Blue Trisacryl M resin in an appropriate storage or sample loading buffer.
8

Sanitization
• Chemical treatments
The following procedures are recommended:
Method
Ethanol – acetic acid washing
Procedure
Wash with at least 3 CV of a solution of
20% ethanol containing 1 M acetic acid.
One hour contact time.
After sanitization, the column must be re-equilibrated in the normal sterile pyrogen-free buffer.
For more information, please contact our technical service.
Storage Recommendations
• The column must be stored at 2-8 °C (36-46 °F) and cannot be frozen.
• Between runs, store the column at 2-8 °C (36-46 °F) in loading buffer.
• The storage buffer may also contain bacteriostatic agents such as 20% (v/v) ethanol and/or 1 M NaCl.
9

Adapter Recommendations
AcroSep pre-packed columns are made with a luer inlet and outlet for easy connection to syringes. The following table lists recommendations if adapters are needed to connect the columns to other types of tubing.
Connection To
1/16" OD Teflon N and Tefzel N Tubing
1/8" OD Teflon and Tefzel Tubing
1/16" Stainless Steel Tubing
1/32" Stainless Steel Tubing
Adapters (Upchurch Scientific N )
1 kit (P-837)
Instructions provided with kit
1 kit (P-838)
Instructions provided with kit
1 inlet fitting (P-658),
1 outlet fitting (P-655),
2 ferrules (P-259), 2 nuts (LT-115)
Instructions provided with fittings
1 inlet fitting (P-658),
1 outlet fitting (P-655),
2 ferrules (P-248), 2 nuts (LT-115)
Instructions provided with fittings
10

WARNING
Employment of the products in applications not specified, or failure to follow all instructions contained in this product information insert, may result in improper functioning of the product, personal injury, or damage to property or the product. See Statement of Warranty in our most recent catalog.
ATTENTION
L’utilisation de nos produits dans des applications pour lesquelles ils ne sont pas spécifiés ou le non-respect du mode d’emploi qui figure sur ce document, peut entrainer un disfonctionnement du produit, endommager le produit ou d’autres biens matériels ou représenter un risque pour l’utilisateur.
Se référer à la clause de garantie de notre catalogue le plus récent.
ACHTUNG
Der Einsatz dieses Produktes in Anwendungen für die es nicht spezifiziert ist, oder das Nichtbeachten einiger, in dieser Bedienungsanleitung gegebenen
Hinweise kann zu einem schlechteren Ergebnis, oder Zerstörung des
Produktes oder anderer Dinge oder gar zu Verletzungen führen. Beachten Sie auch unsere Garantiebedingungen im aktuellen Katalog.
ADVERTENCIA
El uso de este producto en aplicaciones no especificadas o el no considerar las instrucciones indicadas en la hoja de información del producto puede ocasionar un mal funcionamiento del producto, daños en las instalaciones o en el producto y riesgo para el personal del laboratorio. Consulte el apartado de Garantía en nuestro último catálogo.
11

ATTENZIONE
L’impiego dei prodotti in applicazioni non specificate, o il mancato rispetto di tutte le istruzioni contenute nel presente bollettino tecnico, potrebbero portare ad un utilizzo improprio del prodotto, ferire gli operatori, o danneggiare le caratteristiche del prodotto stesso. Consultare la dichiarazione di garanzia pubblicata nel nostro più recente catalogo.
12

Pall Life Sciences
600 South Wagner Road
Ann Arbor, MI 48103-9019 USA
800.521.1520
USA and Canada
(+)800.PALL.LIFE
Outside USA and Canada
734.665.0651
734.913.6114
phone fax
Visit us on the Web at www.pall.com/lab
E-mail us at LabCustomerSupport@pall.com
Pall, , AcroSep, and Trisacryl are trademarks of
Pall Corporation. ® indicates a trademark registered in the USA.
N
ÄKTAdesign is a trademark of GE Healthcare.
Cibacron is a trademark of CIBA-GEIGY Ltd. Upchurch is a trademark of Idex Corporation. Teflon and Tefzel are trademarks of DuPont.
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