HPLC • SMB • Osmometry Affi nity chromatography (AC) purifi cation of specifi c proteins like 2 BioFox 40/1200 ACT and antibodies BioFox 40/10000 ACT Analytical and preparative separations of proteins Pre-activated media for preparative and bioprocess scale affi nity chromatography with user’s choice of ligands sample loading separation elution Y Y Y Y Y Y Y Y Y Y Y Y Affi nity chromatography separates are coupled via their reactive functional proteins on the basis of a reversible groups such as amino, carboxyl, hydroxyl interaction between a protein and a of thiol moieties to the gel. In particular, specifi c ligand covalently bound to a antigens or antibodies as ligands create a chromatographic medium. Thus, the highly selective media for immunoaffi n-typical affi nity chromatography is often ity purifi cation. After loading the protein referred to as ”lock and key principle“ of a mixture into the separation column, part of a molecule (key) with an immobi-non-binding molecules are almost en-lized ligand (lock). Antibodies, antigens, tirely eliminated using rinse buffers, so enzymes, short nucleic acids or peptides that the fi nal elution fraction contains can be used as affi nity ligands. They the target molecule in high purity. Pressure stability up to 40 bar (580 psi) – fast and high resolution biochromatography BioFox 40 ACT pre-activated separation ACT is pressure resistant up to 40 bar tion method is, although proprietary, media is produced from agarose using (580 psi) for high throughput biochro-based upon well-understood chemistry, a proprietary cross-linking method that matography. allowing you to perform the coupling results in a highly porous and physi-reaction in an aqueous solution. cally stable matrix. Besides the well-BioFox 40 ACT is activated according known selectivity of agarose, BioFox 40 to the bromhydrin method. This activa-Step 1: Preparation of an immunoadsorbent with BioFox 40 ACT Matrix-OCH2CH(OH)CH2Br + Nu- (e.g. –SH, –NH2 or –OH) –––> Matrix-OCH2CH(OH)CH2-Nu (Nu = Nucleophiles) Step 2: Coupling reaction in aqueous solution Since no toxic chemicals are necessary BioFox 40 ACT • Suitable for coupling of ligands con-for the coupling procedure and the taining sulfhydryl, amino or hydroxyl BioFox ACT products are stable at room • Made from agarose, well-established groups temperature, coupling can easily be and well-known in the biotechnology • Optimum particle size of 40 µm and performed on your bench and at room industry narrow particle size distribution for temperature. • Simple coupling procedures at room high throughput preparative and bio-temperature process scale applications • Stable at room temperature in aqueous solution and at neutral pH Coupling conditions and selection of Hydroxyl groups can also be used for Stability coupling buffers coupling, but will require pH 12 which is not compatible with most proteins. BioFox 40 ACT media is stable for 12 BioFox 40 ACT comes ready to use. However, stable molecules can be months in aqueous solutions containing Proteins or other biomolecules with coupled using the hydroxyl group. 22 % ethanol at neutral pH and at room free amino and thiol groups will easily Remaining reactive groups are deacti-temperature without any signifi cant de-couple to BioFox 40 ACT. Just add the vated using 2-mercaptoethanol or crease of coupling activity. The choice of ligand to the suspension, stir and ethanolamine. storage buffer for a coupled gel medium incubate overnight. depends on the properties of the ligand. Type of ligand Functional group of ligand Coupling buffers pH 7 and higher. Sensitive ligands Organic molecules, can be coupled at pH 7 but a better peptides Thiol-SH yield is achievable at higher pH. Basicity of the ligand will determine the coupling pH. When the ligand is used in excess, Organic molecules, Amines: primary (-NH2) dissolve the ligand in distilled water peptides secondary (-NHR) tertiary (-NR and the basicity of the ligand will 3) determine the coupling pH. Proteins, pH 7 and higher. Sensitive ligands polypeptides Thiol (-SH) can be coupled at pH 7 but a better yield is achievable at higher pH. Coupling yield will increase at higher pH. A carbonate buffer of pH 8 to 8.5 Proteins, Primary amines (-NH2) often gives suffi cient coupling without polypeptides denaturation of sensitive polypeptides and proteins. Another possibility is to run the coupling reaction at lower temperature. The low nucleophilicity of the hydroxyl group requires coupling All types Hydroxyl (-OH) conditions at very high pH > 12. At a pH < 12 cross-linking and hydrolysis will compete with the coupling procedure. The Bioline Rack – with glass columns HPLC • SMB • Osmometry Media characteristics BioFox 40/1200 ACT Exclusion limit 1 200 kDa Max flow rate at 20 cm bed height > 500 cm/h Particle size 32 – 60 µm Spacer arms 4 – 16 # Atoms Agarose content 7.4 – 7.8 % Coupling groups -OH, -NH2, -SH Degree of substitution 0.6 – 0.7 mol/mol Solvent stability 100 % methanol, 100 % ethanol, 8 M urea, 6 M guanidine hydrochloride, 30 % acetonitrile, 70 % formic acid, 30 % trifluoroacetic acid Media characteristics BioFox 40/10000 ACT Exclusion limit 10 000 kDa Particle size 32 – 60 µm Spacer arms 4 – 16 # Atoms Agarose content 4.6 – 5.0 % Coupling groups -OH, -NH2, -SH Degree of substitution 0.6 – 0.7 mol/mol Solvent stability 100 % methanol, 100 % ethanol, 8 M urea, 6 M guanidine hydrochloride, 30 % acetonitrile, 70 % formic acid, 30 % trifluoroacetic acid BioFox 40/1200 ACT and BioFox 40/10000 ACT media are supplied as an aqueous suspension with 22 % ethanol as a preservative. After washing the media are immediately ready for use. Ordering information BioFox 40/1200 ACT Ordering information BioFox 40/10000 ACT Order No. Volume Order No. Volume 00JX40MBFZ Bulk media, 50 ml 00JX40NBFZ Bulk media, 50 ml 00OX40MBFZ Bulk media, 300 ml 00OX40NBFZ Bulk media, 300 ml 00QX40MBFZ Bulk media, 1 l 00QX40NBFZ Bulk media, 1 l 00RX40MBFZ Bulk media, 5 l 00RX40NBFZ Bulk media, 5 l Technical data are subject to change without notice. Please check our website for latest updates and changes. Visit www.knauer.net for more information on KNAUER’s complete range of biochromatography products. Wissenschaftliche Gerätebau Your local distributor: Dr. Ing. Herbert Knauer GmbH Hegauer Weg 38 14163 Berlin, Germany Phone: +49 30 809727-0 Telefax: +49 30 8015010 E-Mail: info@knauer.net Internet: www.knauer.net © KNAUER 2014 V7836-1US/3.0/02.14/4T