GE HEALTHCARE AKTAprime plus Quick Reference Instructions

Also see for AKTAprime plus: Operating instructions

Contents

GE HEALTHCARE AKTAprime plus Quick Reference Instructions Manual pdf 20 page image

11-0027-48 AC, 2007-09 • p20MBP-tagged protein purification1 Preparing the buffers• Use high purity water and chemicals.• Filter all buffers through a 0.45 μm filter before use.Binding buffer (port A1):20 mM Tris-HCI, 200 mM NaCl, 1 mM EDTA, 1 mM DTT,pH 7.4Elution buffer (port B):20 mM Tris-HCI, 200 mM NaCl, 1 mM EDTA, 1 mM DTT,10 mM maltose, pH 7.4Prepare at least 500 ml of each eluent.2 Preparing the samplea) Adjust the sample to composition of binding buffer by:• diluting the sample in binding buffer or• by buffer exchange using HiTrap Desalting or HiPrep26/10 Desalting.b) Pass the sample through a 0.45 μm filter.3 Preparing the systema) Place the inlet tubing from port A1 (8-port valve)in the binding buffer and the tubing from port B(2-port valve) in the elution buffer.b) Place the three brown waste tubings in waste.c) Connect the column between port 1 on the injectionvalve (7-port valve) and the UV flow cell (see Orderinginformation on next page for suitable columns).d) Fill the fraction collector rack with 18 mm tubes*(minimum 10) and position the white plate on thefractionation arm against the first tube.e) Connect a sample loop large enough for your samplebetween port 2 and 6 on the injection valve. Use asyringe to manually fill the loop.Note: If a Superloop is needed, additional informationis supplied in the instructions for Superloop.* The number of tubes to insert in the fraction collector varies withthe sample volume. Fill the fraction collector with 20 tubes +one tube/ml sample. For example, if the sample volume is 10 ml,fill the fraction collector with 20 + 10 = 30 tubes. However, notethat the maximum capacity of the fraction collector is 95 tubes,limiting the sample volume to 75 ml.4 Selecting Application Template andstarting the methoda) Check the communication to PrimeView. At the lowerright corner of the screen the text Controlled By:prime should be displayed.b) Use the arrow and OK buttons to move in the menutree until you find Affinity Purification any HiTrap.c) Enter the sample volume and press OK to start thetemplate.Note: If a 5 ml column is preferred, see cue card onp.36.Theoretical gradient in Affinity Purification any HiTrap ApplicationTemplate.1 10 20 10 5 Min10050SamplePrimingElutionTotal separation time = 47 min + sample application timeWater wash &priming%BRe-equili-brationEquilibrationAffinity Purificationany HiTrapSet Sample Inj. Vol(00.0 ml) 00.0Run Application TemplatePress OK to startRun data displayedApplication TemplateTemplates