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instructions

HiTrap Protein A HP, 1 ml and 5 ml


HiTrap affinity columns
HiTrapTM Protein A HP is a prepacked ready to use, column for preparative purification of monoclonal and polyclonal antibodies. The special design of the column, together with the matrix provide 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 KTAdesign or FPLC System.

i 71-7002-00 Edition AK

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Code No. 17-0402-01 17-0402-03 17-0403-01

Designation HiTrap Protein A HP HiTrap Protein A HP HiTrap Protein A HP Connectors Luerlock female/M6 male Luerlock female/M6 female Tubing connector flangeless/M6 male Tubing connector flangeless/M6 female Union M6 female/1/16 male Union 1/16 female/M6 male Domed nut Instructions

No. supplied 5 x 1 ml 2 x 1 ml 1 x 5 ml 1 1 1 1 1 1 2 or 5 1

Description
Column HiTrap Protein A HP 1 ml and 5 ml columns are made of polypropylene, which is biocompatible and non-interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene. 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 KTA or FPLC. Note: To prevent leakage it is essential to ensure that the adaptor is tight.

The column cannot be opened or refilled.

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Gel properties HiTrap Protein A HP 1 ml and 5 ml columns are packed with 1 ml and 5 ml of Protein A SepharoseTM High Performance, respectively. Protein A Sepharose High Performance is designed for purification and isolation of monoclonal and polyclonal IgG from ascites, serum and cell culture supernatants (Table 2). Protein A is produced by a selected strain of Staphylococcus aureus and has a molecular weight of 42 000. Protein A consists of six different regions, five of which show strong, specific binding for the Fc-part of IgG, leaving the antigen-binding sites free. Immobilized protein A can bind at least two molecules of IgG per molecule. In general, one can purify most IgGs using protein A. A reference list showing the use of HiTrap Protein A HP columns (Code No. 18-1156-73) is available, visit www.chromatography.amershambiosciences.com. Purified protein A is coupled to highly cross-linked agarose beads by the N-hydroxysuccinimide activation method. This coupling method gives high capacity and high performance. The binding capacity of protein A for IgG depends on the source species of the particular immunoglobulin. The total capacity depends also upon several other factors such as the flow rate during sample application, and the sample concentration. This gel has a binding capacity for human IgG of approximately 20 mg IgG/ml gel.

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The characteristics of HiTrap Protein A HP are summarized in Table 1.


Table 1. HiTrap Protein A HP characteristics
Column volumes Column dimensions Ligand Degree of substitution Binding capacity Mean particle size Max. back pressure Bead structure Max. flow rates columns respectively Rec. flow rates Chemical stability pH stability* Long term Short term Storage buffer 1 ml and 5 ml 0.7 x 2.5 cm (1 ml) and 1.6 x 2.5 cm (5 ml) Protein A, Mr 42 000 ~3 mg protein A/ml gel ~20 mg human IgG/ml gel 34 m 0.3 MPa, 3 bar Highly cross-linked spherical agarose 4 and 20 ml/min for 1 and 5 ml 1 ml/min and 5 ml/min for 1 ml and 5 ml column respectively All commonly used buffers 39 2 10 20% ethanol

* The ranges given are estimates based on our knowledge and experience. Please note the following: pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. pH below 3 is sometimes reqired to elute strongly bound IgG species. However, protein ligands may hydrolyze at very low pH.

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Table 2. Relative binding strength of polyclonal IgG from various species to protein G and protein A.
Species Human IgG1 IgG2 IgG3 IgG4 Rabbit Cow Horse Goat Guinea pig Sheep Dog Pig Rat* Mouse** Chicken Protein G ++ ++ ++ ++ ++ ++ ++ ++ + ++ + ++ + Protein A ++ ++ ++ ++ + + ++ ++ ++ (+) -

Binding is measured in a competitive ELISA test. The amount of IgG required to give a 50% inhibition of binding of rabbit IgG conjugated with alkaline phosphatase was determined. ++ = strong bindning + = medium binding - = weak or no binding
* Note that IgG from rat binds to protein G when it is attached to a matrix. ** IgG1 from mouse binds stronger to protein G than to protein A

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Operation
Protein A binds IgG over a wide pH range, and thus permits the use of a wide variety of buffers, depending on the applications. Elution is often achieved by a decrease in pH. Different subclasses of IgG elute at different pH values depending on the species from which they originate. As a safety measure to preserve the activity of acid labile IgG when using very acidic elution conditions, we recommend you to add 60-200 l of 1 M Tris-HCl, pH 9.0, to those tubes destined to collect fractions containing IgG, so that the final pH of the sample will be approximately neutral. 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 m filter before use. Recommended buffers: Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M citric acid, pH 3-6

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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, see Table 2 or using a HiPrep 26/10 Desalting column. The sample should be filtered through a 0.45 m filter or centrifuged immediately before it is applied to the column. (This is especially important to prevent clogging of the column when loading large volumns of serum or plasma.)

Purification
1. Prepare collection tubes by adding 60-200 l of 1 M Tris-HCl pH 9.0 per ml of fraction to be collected. 2. 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. 3. Remove the twist-off end. 4. Wash the column with 10 column volumes of binding buffer at 1 ml/min or 5 ml/min for 1 ml and 5 ml column respectively. 5. Apply the sample, using a syringe fitted to the luer adaptor or by pumping it onto the column. 6. Wash with 5-10 column volumes of binding buffer or until no material appears in the effluent. 7. Elute with 2-5 column volumes of elution buffer. 8. The purified fractions can be buffer exchanged using HiTrap Desalting or PD-10 column if necessary.
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Note:

The reuse of HiTrap Protein A HP depends on the nature of the sample and should only be performed with identical antibodies to prevent cross-contamination. If a P-1 pump is used a max flow rate of 1-3 ml/min can be run on a HiTrap 1 ml column packed with Sepharose HP media.

Note:

Scaling up
For quick scale-up of purification, two or three HiTrap columns can be connected in series (backpressure will increase). If further scale-up is necessary bulk media is available.

Storage
Before storage, we recommend to wash the column with 5 column volumes of 20% ethanol to prevent microbial growth. Store the column in 20% ethanol at +4 to +8 oC.

Furher information
Visit www.chromatography.amershambiosciences.com for more information. Useful handbooks are available from Amersham Biosciences, see ordering information.

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p10
Column HiTrap Desalting 0.1 1.5 ml 1.3 4.0 ml x applied volume 3.5 ml Prepacked with Sephadex G-25. Requires only gravity to run. Prepacked with Sephadex G-50 DNA grade. Requires only gravity to run. Prepacked with SephadexTM G-25 Superfine. Requires a syringe or a pump to run. Load Elute Comments Notes For desalting and buffer exchange of protein extracts (Mr >5000). PD-10 2.5 ml For desalting and buffer exchange of protein extracts (Mr >5000). For separation of proteins (Mr >30000) and nicktranslated DNA from radiolabeled nucleotides no shorter than 120 mers and similar separations. For purification of proteins, (Mr >5000) DNA and oligonucleo-tides greater than 10 bases in length. NICK TM 0.1 ml 0.4 ml NAP TM -5 NAP-10 NAP-25 0.5 ml 1.0 ml 2.5 ml 1.0 ml 1.5 ml 3.5 ml Prepacked with Sephadex G-25 DNA grade. Requires only gravity to run.

Table 2. Prepacked columns for desalting & buffer exchanges.

Code No.

17-1408-01

17-0851-01

17-0855-01

17-0853-01 17-0854-01 17-0852-01

Ordering Information
Designation HiTrap Protein A HP, 1 ml HiTrap Protein A HP, 1 ml HiTrap Protein A HP, 5 ml HiTrap rProtein A FF, 1 ml HiTrap rProtein A FF, 1 ml HiTrap rProtein A FF, 5 ml HiTrap Protein G HP, 1 ml HiTrap Protein G HP, 1 ml HiTrap Protein G HP, 5 ml MAbTrapTM Kit HiTrap Desalting, 5 ml PD-10 Disposible Column NICK Columns NAP-5 Columns NAP-10 Columns NAP-25 Columns No. Supplied 2 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 2 x 1 ml 5 x 1 ml 1 x 5 ml 1 kit 5 x 5 ml 30 20 20 20 20 4 2 2 2 2 5 6 1 1 1 1 Code No. 17-0402-03 17-0402-01 17-0403-01 17-5079-02 17-5079-01 17-5080-01 17-0404-03 17-0404-01 17-0405-01 17-1128-01 17-1408-01 17-0851-01 17-0855-01 17-0853-01 17-0854-01 17-0852-01 18-2450-01 18-1027-12 18-1027-62 18-1003-68 18-1017-98 18-3858-01 18-1112-57 18-1037-46 18-1022-29 18-1121-86 18-1129-81

Accessories Domed nut* Union Luerlock female/M6 female* female/M6 male* Tubing connector flangeless/M6 female* flangeless/M6 male* To connect columns with M6 connections to KTAdesign Union M6 female /1/16" male* Union 1/16 female/M6 male* Antibody Purification Handbook Affinity Chromatography Handbook, Principles and Methods Affinity Chromatography Column and Media Selection Guide Convenient Protein Purification, HiTrap Column Guide * included in HiTrap package

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Amersham Biosciences AB Bjrkgatan 30 SE-751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place, Little Chalfont Buckinghamshire HP7 9NA England Amersham Biosciences Corp. 800 Centennial Avenue PO Box 1327 Piscataway, NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111 Freiburg Germany Amersham Biosciences K.K. Sanken Building, 3-25-1 Shinjuku-ku, Tokyo 169-0073 Japan Visit us at:

www.chromatography.amershambiosciences.com
Trademarks
HiTrap, HiPrep, Sepharose, Sephadex, FPLC, KTA, MAbTrap, NICK and NAP are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. Drop Design is a trademark of Amersham Biosciences Limited. Amersham Biosciences AB 2002 All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request.