HiTrap Affinity columns

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1 HiTrap Affinity columns HiTrap Peanut Lectin, 1 ml INSTRUCTIONS HiTrap TM Peanut Lectin is a pre-packed, ready-to-use, disposable column for preparative affinity chromatography. The special design of the column, together with the matrix, provides fast, simple and easy separations in a convenient format. The column can be operated with a syringe, peristaltic pump or liquid chromatography system such as ÄKTA TM, GradiFrac TM or FPLC TM. Table 1. Content of HiTrap Peanut Lectin Code No. Designation No. supplied HiTrap Peanut Lectin 5x1 ml Connectors Luerlock female/m6 male 1 Luerlock female/m6 female 1 Tubing connector flangeless/m6 male 1 Tubing connector flangeless/m6 female 1 Domed nut 5 Instructions Edition AC

2 Description Gel properties Peanut Lectin (PNA) is isolated from Arachis hypogaea and coupled to NHS-activated Sepharose TM High Performance. The base matrix is a rigid, highly cross-linked, beaded agarose with high chemical stability. The specificity of Peanut Lectin is show in the table below. Table 2. Specificity of lectins Lectin Mannose/glucose binding lectins Con A, Canavalia ensiformis Lentil Lectin, Lens culinaris Specificity Branched mannoses, carbohydrates with terminal mannose or glucose (αman>αglc>glcnac) Branched mannoses with fucose linked α(1,6) to the - N-acetyl-glucosamine, (αman>αglc>glcnac) N-acetylglucosamine binding lectins Wheat Germ Lectin, Triticum vulgare Chitobiose core of N- linked oligosaccharides, [GlcNAc(β1,4GlcNAc) 1-2 > βglcnac] N-acetylgalactosamine/galactose binding lectins Peanut Lectin, Arachis hypogaea Terminal β-galactose, (Galβ1,3GalNAc> α and βgal) 2

3 Peanut Lectin is a tetrameric, carbohydrate-free protein and it has selectivity for terminal β-galactose and recognizes the T- antigen (Galβ1,3GalNAc). The subunits contain one atom each of Ca 2+ and Mg 2+. Application areas for HiTrap Peanut Lectin include purification of glycoproteins such as the Gal(β1,3)GalNAc bearing glycoproteins at the nodes of Ranvier in peripheral nerve (Ref. 1), natural human antibodies to dietary lectins (Ref. 2), and peanut agglutinin-binding glycoproteins from lizard lymphocytes (Ref. 3). Other applications include the characterization of carbohydrate moieties of glycoproteins such as CBP70 isolated from HL60 cells (Ref. 4), and PCA inhibitory factor present in mouse serum (Ref. 5). Column The column is made of medical grade poly-propylene, which is biocompatible and non-interactive with biomolecules. The column has porous polyethylene top and bottom frits. It is delivered with a stopper on the inlet and a twist-off end on the outlet. Both ends have M6 connections (6 mm metric threads). The separation can be easily achieved using a syringe together with the supplied luer adaptor, a peristaltic pump, or in a chromatography system such as ÄKTA, GradiFrac, or FPLC. Note: To prevent leakage it is essential to ensure that the adaptor is tight. The column cannot be opened or refilled. The characteristics of the product are summarised below in Table 3. 3

4 Table 3. HiTrap Peanut Lectin characteristics Column dimensions i.d. x h 0.7 x 2.5 cm Column volume 1 ml Ligand Peanut Lectin Ligand concentration 4 mg/ml Binding capacity 3 mg Asialofetuin/ml Mean particle size 34 µm Bead structure Highly cross-linked spherical agarose Maximum back pressure 0.3 MPa, (3 bar) Maximum flow rate 4 ml/min Recommended flow rate ml/min ph stability 4-9 Temperature stability Regular use 4 C - room temperature Storage 4-8 C Storage buffer Binding buffer with 20 % ethanol Operation The column can be operated with a syringe, peristaltic pump or a chromatography system. Buffer preparation Water and chemicals used for buffer preparation should be of high purity. It is recommended to filter the buffers by passing them through a 0.45 µm filter before use. Binding of glycoproteins occurs at neutral ph. Recommended buffers: Binding buffer: 10 mm sodium phosphate, 0.15 M NaCl, 0.5 mm MgCl 2, 0.5 mm CaCl 2, ph 7.4. Elution buffer: 0.5 M β-lactose, 10 mm sodium phosphate, 0.15 M NaCl, 0.5 mm MgCl 2, 0.5 mm CaCl 2, ph

5 Sample preparation The sample should be adjusted to the composition of the binding buffer. This can be done by either diluting the sample with binding buffer or by buffer exchange using HiTrap Desalting or PD-10 column. The sample should be filtered through a 0.45 µm filter or centrifuged before it is applied to the column. Purification The recommended flow rate is 1 ml/min, unless other flow rate is stipulated. Note: 1 ml/min corresponds to approximately 30 drops/min, when the column is operated with a syringe. 1. Fill the syringe or pump tubing with binding buffer. Remove the stopper and connect the column to the syringe (with the provided adaptor), or pump tubing, drop to drop to avoid introducing air into the column. 2. Remove the twist-off end. 3. Wash and equilibrate the column with 10 column volumes of binding buffer. 4. Apply the sample, using a syringe fitted to the luer adaptor or by pumping it onto the column. For best results use a low flow rate, ml/min, during sample application. 5. Wash with 5-10 column volumes of binding buffer or until no material appears in the effluent. 6. Elute with 5-10 column volumes of elution buffer. 5

6 Note: Before reuse the column has to be regenerated by washing with 10 column volumes of 1 M NaCl followed by reequilibration with at least 25 column volumes of binding buffer. The reuse of HiTrap Peanut Lectin depends on the nature of the sample and should only be performed with identical samples to prevent cross-contamination. Binding The most important parameter affecting the binding of glycoproteins to the matrix, is the flow rate. It is important to keep the flow rate as low as ml/min during sample application for maximum binding capacity. When applying the sample with a syringe an alternative to keeping the flow rate low is to apply 1 ml at the time and let it bind for a couple of minutes. To preserve binding activity excess Mg 2+ and Ca 2+ (0.5 mm) must be present. Elution The recovery of glycoproteins can sometimes be improved by pausing the flow for a few minutes during elution. For complex samples containing glycoproteins with different affinity for the lectin, a continuous gradient of M β- lactose, or a stepgradient of galactose followed by β-lactose is recommended. A continuous gradient can be achieved by use of a chromatography system, such as GradiFrac, FPLC, ÄKTA, or by use of a pump and a gradient mixer. 6

7 Storage Store the column at 4-8 C in binding buffer with 20 % ethanol. References 1. Identification of Gal(b1-3)GalNAc bearing glycoproteins at the nodes of Ranvier in peripheral nerve. J. Neurosci. Res. 38 (1994) Apostolski S, Sadiq SA, Hays A, Corbo M, Suturkova-Milosevic L, Chaliff P, Stefansson K, LeBaron RG, Ruoslahti E, Hays AP and Latov N. 2. Natural human antibodies to dietary lectins. FEBS Lett. 397 (1996) Tchernychev B and Wilchek M. 3. Identification of peanut agglutinin-binding glycoproteins on lizard lymphocytes. Zoolog. Sci. 12 (1995) Mansour MH, Negm HI, Saad AH, Zahran AY, and Badir N. 4. CBP70, a glycosylated nuclear lectin. J. Cell. Biochem. 66 (1997) Rousseau C, Felin M, Doynette-Moyne M-A and Seve A-P. 5. An anaphylaxis inhibitory factor present in mouse serum. J. Investig. Allergol. Clin. Immunol. 6 (1996) Astorquiza MI and Droguett MA. 7

8 Ordering Information Designation No. Supplied Code No. GSTrap TM, 1 ml 5x1 ml GSTrap, 1 ml 2x1 ml GSTrap, 5 ml 1x5 ml HiTrap Peanut Lectin, 1 ml 5x1 ml HiTrap Con A, 1 ml 5x1 ml HiTrap Lentil Lectin, 1 ml 5x1 ml HiTrap Wheat Germ Lectin,1ml 5x1 ml HiTrap Lectin Test Kit, 1 ml 4x1 ml HiTrap Blue, 1 ml 5x1 ml HiTrap Blue, 5 ml 1x5 ml HiTrap NHS-activated, 1 ml 5x1 ml HiTrap NHS-activated, 5 ml 1x5 ml HiTrap Desalting, 5 ml 5x5 ml HiTrap SP, 1 ml 5x1 ml HiTrap SP, 5 ml 5x5 ml HiTrap Q, 1 ml 5x1 ml HiTrap Q, 5 ml 5x5 ml HiTrap IEX test kit, 1 ml 4x1 ml HiTrap rprotein A, 1 ml 2x1 ml HiTrap rprotein A, 1 ml 5x1 ml HiTrap rprotein A, 5 ml 1x5 ml HiTrap Protein A, 1 ml 2x1 ml HiTrap Protein A, 1 ml 5x1 ml HiTrap Protein A, 5 ml 1x5 ml HiTrap Protein G, 1 ml 2x1 ml HiTrap Protein G, 1 ml 5x1 ml HiTrap Protein G, 5 ml 1x5 ml MAbTrap TM G II kit 1 kit HiTrap Heparin, 1 ml 5x1 ml HiTrap Heparin, 5 ml 1x5 ml HiTrap Chelating, 1 ml 5x1 ml HiTrap Chelating, 5 ml 1x5 ml HiTrap IgM Purification, 1 ml 5x1 ml HiTrap IgY Purification, 5 ml 1x5 ml HiTrap Streptavidin, 1 ml 5x1 ml HisTrap TM 1 kit HiTrap HIC Test Kit, 1 ml 1 kit PD-10 Disposible Column

9 Accessories Designation No. Supplied Code No. Domed nut* Union Luerlock female/m6 female* female/m6 male* Tubing connector flangeless/m6 female* flangeless/m6 male* Union female /1/16"male (to connect columns with M6 connections to ÄKTAdesign) * included in HiTrap package 9

10 Important Information HiTrap, Sepharose, FPLC, ÄKTA, HisTrap, GSTrap, MAbTrap and GradiFrac are trademarks of Amersham Biosciences Limited or its subsidiaries. Amersham and Amersham Biosciences is a trademark of Amersham plc. Amersham Biosciences AB All rights reserved Amersham Biosciences AB SE Uppsala Sweden Amersham Biosciences UK Limited Amersham Place, Little Chalfont Buckinghamshire HP7 9NA England Amersham Biosciences Inc P.O. Box 1327 Piscataway, NJ USA Amersham Biosciences Europe GmbH Postfach 5480 D Freiburg Germany 10