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            <ITEXT CH="Protein Purification with the Äkta Avant"/>
            <para PARENT="Title"/>
            <ITEXT CH="© Enni Isokangas, Michael Jeltsch &amp; University of Helsinki"/>
            <para PARENT="Heading2" ALIGN="1"/>
            <para PARENT="Heading2" ALIGN="1"/>
            <ITEXT CH="This guide describes how to use the Äkta Avant 25, which is available for protein purification and located in the Protein Production and Purification Core Facilty ("/>
            <ITEXT FCOLOR="Blue" CH="http://research.med.helsinki.fi/corefacilities/akta/index.html)"/>
            <ITEXT CH=". This manual is a work in progress and if you find anything missing, please let us know. This manual does not go into theory at all, but only describes the steps needed to perform a two step purification. In this guide, three often used examples of affinity chromatography methods are described: Ni"/>
            <ITEXT FEATURES="inherit superscript" CH="2+"/>
            <ITEXT CH="NTA affinity chromatography, Protein A affinity chromatography and gel filtration (also called size exclusion chromatography; desalting = gel filtration with low resolution that only separates low molecular weight constituents like salts and amino acids from proteins, but doesn't separate proteins by their size). In our examples, we purify secreted proteins from conditioned cell culture supernatant. Because we have a rather large sample volume (400 ml), the sample is loaded directly to the column with the sample pump in the first (affinity chromatography) step. The second step is the size exclusion chromatography/gel filtration. In this step, the sample (the peak of choice from the elution of the first chromatography step) is loaded with a syringe into the capillary loop, from which it is injected into the gel filtration column. "/>
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            <ITEXT CH="1. Buffers needed"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="For Ni2+NTA affinity chromatography"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT CH="∙ Ni2+NTA binding buffer: 30 mM phosphate buffer pH 8.0, 400 mM NaCl. How much? ≥250 ml for a 1-ml-column, ≥500 ml for a 5-ml-column "/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" Ni"/>
            <ITEXT FEATURES="inherit superscript" CH="2+"/>
            <ITEXT CH="NTA elution buffer: 30 mM phosphate buffer pH 8.0, 400 mM NaCl, 250 mM imidazole. How much? ≥100 ml for a 1-ml-column, ≥250 ml for a 5-ml-column "/>
            <para PARENT="List"/>
            <tab FONT="Arial Regular"/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" column storage buffer: 20% ethanol"/>
            <para PARENT="List"/>
            <ITEXT CH="For Protein A affinity chromatography"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" PBS-T (0.1% Tween-20). How much? ≥250 ml for a 1-ml-column, ≥500 ml for a 5-ml-column "/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" PAS elution buffer: 0.1 M citrate pH 3.0. How much? ≥100 ml for a 1-ml-column, ≥250 ml for a 5-ml-column "/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" column storage buffer: 20% ethanol"/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" neutralization buffer: 1M Tris/HCl, pH 8.5 How much? 5-10 ml."/>
            <para PARENT="List"/>
            <ITEXT CH="For Gel Filtration chromatography"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" Any buffer that is compatible with the protein you want to purify. PBS, TBS or HBS are commonly used buffers. How much? Depending on the column volume: For 10/100 columns (10/100 = 10 mm inner diameter, 100 mm column height): ≥100ml, for 10/300  columns: ≥250 ml, for 16/600 columns: ≥1 liter, for 26/600 columns: ≥2 liter"/>
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            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" column storage buffer: 20% ethanol"/>
            <para PARENT="List"/>
            <ITEXT CH="2. Columns needed"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="For Ni2+NTA affinity chromatography"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT CH="Depending on the amount of protein that you want to purify:"/>
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            <tab/>
            <ITEXT CH="HisTrap FF 1 ml (GE Healthcare, 17-5319-01) for up to 40 mg of protein; HisTrap FF 5 ml (GE Healthcare, 17-5255-01 ) for more than 40 mg. Alternative columns for special cases are: HisTrap FF Crude (for purification from cell lysate), HisTrap HP (higher binding capacity at same column dimensions), HisTrap Excel (chemically more stable and higher affinity for his-tagged proteins, but only about 25% binding capacity)"/>
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            <ITEXT CH="For Protein A affinity chromatography"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT CH="Depending on the amount of protein that you want to purify:"/>
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            <ITEXT CH="HiTrap rProtein A FF 1 ml (GE Healthcare, 17-5079-01) for up to 40 mg of human IgG"/>
            <ITEXT FEATURES="inherit subscript" CH="1"/>
            <ITEXT CH="; HiTrap rProtein A FF 5 ml(GE Healthcare, 17-5080-01) for more than 40 mg of human IgG"/>
            <ITEXT FEATURES="inherit subscript" CH="1"/>
            <ITEXT CH=" "/>
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            <ITEXT CH="For Gel filtration/Desalting"/>
            <para PARENT="Heading2"/>
            <tab/>
            <ITEXT CH="Depending on the volume of protein that you want to purify/desalt, you need differently sized columns:"/>
            <tab/>
            <tab/>
            <tab/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT FONT="Arial Bold Italic" CH="Column  "/>
            <tab FONT="Arial Bold Italic"/>
            <tab FONT="Arial Bold Italic"/>
            <tab FONT="Arial Bold Italic"/>
            <tab FONT="Arial Bold Italic"/>
            <tab FONT="Arial Bold Italic"/>
            <ITEXT FONT="Arial Bold Italic" CH="max. sample volume"/>
            <para PARENT="List"/>
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            <ITEXT CH="10/300 Superdex 200 (Increase)"/>
            <tab/>
            <tab/>
            <tab/>
            <ITEXT CH="1 ml"/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="HighLoad 16/600* or 16/60*"/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <tab/>
            <ITEXT CH="2 ml"/>
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            <tab/>
            <ITEXT CH="HighLoad 26/600* or 26/60*"/>
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            <tab/>
            <ITEXT CH="5 ml "/>
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            <tab/>
            <ITEXT CH="FastDesalting HR 10/10*"/>
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            <tab/>
            <ITEXT CH="1 ml"/>
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            <ITEXT CH="3. Reservation, logging in and starting the program"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="3.1"/>
            <tab/>
            <ITEXT CH="Do not forget to reserve the machine at http://jeltsch.akta.org/ → 'Online reservation system'. You need to log in with your University login/password in order to make a reservation. Please leave your phone number in case we need to contact you."/>
            <para PARENT="List"/>
            <ITEXT CH="3.2"/>
            <tab/>
            <ITEXT CH="Switch on the Äkta Avant and the computer if they are not running already. It does not matter in which order you switch them on. The Äkta ON/OFF switch is on the left side of the machine, near the bottom edge:"/>
            <para PARENT="List"/>
            <ITEXT CH="3.3"/>
            <tab/>
            <ITEXT CH="You need to login twice: First into the computer and then into the UNICORN program. To login into the computer, you may use either your own university account or if more than one person need to control the FPLC device, you can login in using a local account (user: Unicorn, pw: see the note attached to the computer). Then start the UNICORN program via the desktop icon. To login to the program, you need to use your own university  account. In the login window, make sure that the Adminstrator box is not checked:"/>
            <para PARENT="List"/>
            <ITEXT CH="3.4"/>
            <tab/>
            <ITEXT CH="The machine should now already connect to the computer. If this doesn't happen automatically (usually the first time you log in) you can connect manually by clicking 'system' → 'Connect to systems' from the Unicorn program."/>
            <para PARENT="List"/>
            <ITEXT CH="4A. Preparation of the system for a run (Affinity chromatography)"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="4A.1"/>
            <tab/>
            <ITEXT CH="Rinse the inlet tubes by dipping them in clean PBS/H"/>
            <ITEXT FEATURES="inherit subscript" CH="2"/>
            <ITEXT CH="O before placing them in your buffer bottles. Put your buffers in their appropriate places and inlet tubes in the right buffer bottles."/>
            <para PARENT="List"/>
            <tab/>
            <tab FONT="Arial Bold Italic" FONTSIZE="12"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="12" CH="Ni2+NTA"/>
            <tab FONT="Arial Bold Italic" FONTSIZE="12"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="12" CH="Protein A"/>
            <para PARENT="Table Paragraph2"/>
            <tab/>
            <ITEXT CH="A1:"/>
            <tab/>
            <ITEXT CH="Ni2+NTA binding buffer"/>
            <tab/>
            <ITEXT CH="PBS-T"/>
            <para PARENT="Table Paragraph2"/>
            <tab/>
            <ITEXT CH="B1:"/>
            <tab/>
            <ITEXT CH="Ni2+NTA elution buffer"/>
            <tab/>
            <ITEXT CH="0.1 M citrate pH 3.0"/>
            <para PARENT="Table Paragraph2"/>
            <tab/>
            <ITEXT CH="B3:"/>
            <tab/>
            <ITEXT CH="column storage buffer"/>
            <tab/>
            <ITEXT CH="column storage buffer"/>
            <para PARENT="Table Paragraph2"/>
            <tab/>
            <ITEXT CH="Buff:"/>
            <tab/>
            <ITEXT CH="Ni2+NTA binding buffer"/>
            <tab/>
            <ITEXT CH="PBS-T"/>
            <para PARENT="Table Paragraph2"/>
            <tab/>
            <ITEXT CH="S4:"/>
            <tab/>
            <ITEXT CH="sample"/>
            <tab/>
            <ITEXT CH="sample"/>
            <para PARENT="Table Paragraph2"/>
            <ITEXT CH="4A.2"/>
            <tab/>
            <ITEXT CH="Place the sample bottle into an ice-filled styrofoam box on top of the Äkta. You can additionally add 0.1% NaN"/>
            <ITEXT FEATURES="inherit subscript" CH="3"/>
            <ITEXT CH=" to the sample in order to prevent microbial growth."/>
            <para PARENT="List"/>
            <ITEXT CH="4A.3"/>
            <tab/>
            <ITEXT CH="Place a bottle into the fridge to collect your flowthrough, connect tubing Out5 to the bottle."/>
            <para PARENT="List"/>
            <ITEXT CH="4A.4"/>
            <tab/>
            <ITEXT CH="Empty the 2-liter waste bottle if needed."/>
            <para PARENT="List"/>
            <ITEXT CH="4B. Preparation of the system for a run (Gel filtration)"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="4B.1"/>
            <tab/>
            <ITEXT CH="Rinse the inlet tubes (e.g. by dipping them in clean H"/>
            <ITEXT FEATURES="inherit subscript" CH="2"/>
            <ITEXT CH="O) before placing them in your buffer bottles. Put your buffers in their appropriate places and inlet tubes in the right buffer bottles."/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="A1:"/>
            <tab/>
            <tab/>
            <tab/>
            <ITEXT CH="Running buffer (PBS, TBS or HBS)"/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="B3:"/>
            <tab/>
            <tab/>
            <tab/>
            <ITEXT CH="20% ethanol"/>
            <para PARENT="List"/>
            <ITEXT CH="4A.2"/>
            <tab/>
            <ITEXT CH="Empty the 2-liter waste bottle if needed."/>
            <para PARENT="List"/>
            <ITEXT CH="5. Priming/purging the pumps"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="5.1"/>
            <tab/>
            <ITEXT CH="If the inlet tubings A1, B1, B3 and Buff (for affinity chromatography) and A1 and B3 (for gel filtration) are NOT filled with buffer, you MUST fille them! Otherwise the pump will run dry and break! To do this you attach a syringe to all 6 pump purge valves and draw about 20 ml buffer."/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="In order to fill inlet tubing B3 and S4, you need to switch from B1 to B3 and from Buf to S3, respectively, using the UNICORN software. For B3: Manual instructions → Flow path → Pump B inlet: choose inlet B3. For S4: Manual instructions → Flow path → Sample inlet: choose inlet S4."/>
            <para PARENT="List"/>
            <ITEXT CH="5.2"/>
            <tab/>
            <ITEXT CH="Inlet tubings A1, B1 and Buff are the default positions of the valves and can be filled without changing valve positions with the UNICORN software. The sample you draw into your syringe while filling tube S4 can be returned into the sample bottle."/>
            <para PARENT="List"/>
            <ITEXT CH="6. Attaching the column"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="6.1"/>
            <tab/>
            <ITEXT CH="Start a flow (1 to 2 ml/min) with the same buffer that has been used for column storage (mostly 20% EtOH):"/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="Pumps and pressure -&gt; System Flow; select for &quot;Pressure control&quot; D column pressure; Execute. You will be prompted to select the correct column from a list. Once you have selected the correct column, the instructions are in the execution window and you still have to click &quot;Execute&quot;! Let the run proceed until all curves are running (nearly) horizontal."/>
            <para PARENT="List"/>
            <tab/>
            <ITEXT CH="Then switch the flow path to the column position that you want to use (see the table below for the column default position for the method you are going to use): Flow Path -&gt; Column position -&gt; Position 1 (or whatever default position your mehthod is using, in the following, we'll use position 1 as an example). Buffer will start to flow out from posiiton 1A of valve V9-C. Let the outflow continue for a few ml in order to clean the valve. Then connect the top of the column to position 1A. Before attaching the new column, fill the air-filled space at the top of the column by dripping buffer on it to remove the air that might be trapped inside. Once you have attached the column, the system will now go into overpressure and pause if you have have not opened the bottom of the column. That is totally fine, nothing to worry about. Just open the bottom of the column and connect it to position 1B of the same valve (V9-C)."/>
            <para PARENT="List"/>
            <ITEXT CH="7. Calibrate the pH monitor (optional)"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="Calibrating the pH monitor is not necessary if you just want to monitor the pH, but don't need absolute pH values. We do it about once a month, but this means that it may be off by 0.1 to 0.4 pH units if you leave it uncalibrated."/>
            <para/>
            <ITEXT CH="8. Preparing the fraction collector"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="Fractions can be collected into many different tube types. The fraction collector holds six cassettes and we have casette types available for the following tubes/plates:"/>
            <para PARENT="Default Paragraph Style"/>
            <tab/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" 4 cassettes for 96-deep-well plates"/>
            <breakline/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" 2 cassettes for 5-ml-tubes (40 tubes/casette)"/>
            <breakline/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" 2 cassettes for 15-ml-tubes (15 tubes/casette)"/>
            <breakline/>
            <ITEXT FONT="Arial Regular" CH="∙"/>
            <ITEXT CH=" 2 cassettes for 50-ml-tubes (6 tubes/cassette)"/>
            <para PARENT="List"/>
            <ITEXT CH="For the Ni2+NTA affinity chromatography, we are using 15 ml Falcon tubes and for the Protein A affinity chromatography, we use Nunc 5 ml 75x12 Minisorp tubes. For the gel filtration, we use 96-deep-well plates with 1-ml-wells or 2-ml-wells. For a typical affinity purification you should place at least 10 tubes into the cassettes starting from position A1. For the Protein A affinity chromatography, you need to pipette 200 µl 1M Tris pH 8.5 into each fraction tube, because you need to neutralize the low pH of the elution buffer as soon as possible after the elution in order to prevent damaging your protein. For the gel filtration, place one or two (depending on your fraction size) 96-deep-well blocks into the 96-well-plate cassette. Always change the casettes only when the system is not running. (When the system is not running, it does a &quot;full scan&quot; and recognizes all cassettes. You can open the fraction collector also during a run, but you cannot swap different types of cassettes as the system will not recgnize the new order of the cassettes, this is called a &quot;quick scan&quot;.)"/>
            <para PARENT="Default Paragraph Style"/>
            <ITEXT CH="9. Methods"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="A description of method creation is coming in a later version of this manual. For routine purifications (including Ni"/>
            <ITEXT FEATURES="inherit superscript" CH="2+"/>
            <ITEXT CH="NTA, Protein A affinity and gel filtration chromatography), there are ready methods available in the Unicorn software, which can be used:"/>
            <para PARENT="Default Paragraph Style"/>
            <ITEXT FONT="Arial Bold" CH="Default methods:"/>
            <para PARENT="Default Paragraph Style"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="11" CH="method name"/>
            <tab FONT="Arial Bold Italic" FONTSIZE="11"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="11" CH="chromatography"/>
            <tab FONT="Arial Bold Italic" FONTSIZE="11"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="11" CH="column"/>
            <tab FONT="Arial Bold Italic" FONTSIZE="11"/>
            <ITEXT FONT="Arial Bold Italic" FONTSIZE="11" CH="column default"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="Ni2+NTA_HisTrap_1ml_default"/>
            <tab/>
            <ITEXT CH="Ni2+NTA affinity "/>
            <tab/>
            <ITEXT CH="1-ml-HisTrap or similar"/>
            <tab/>
            <ITEXT CH="position 5"/>
            <tab/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="Ni2+NTA_HisTrap_5ml_default"/>
            <tab/>
            <ITEXT CH="Ni2+NTA affinity"/>
            <tab/>
            <ITEXT CH="5-ml-HisTrap or similar"/>
            <tab/>
            <ITEXT CH="position 5"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="HiTrapPAS_1ml_default"/>
            <tab/>
            <ITEXT CH="Protein A affinity"/>
            <tab/>
            <ITEXT CH="1-ml-PAS or similar"/>
            <tab/>
            <ITEXT CH="position 5"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="HiTrapPAS_5ml_default"/>
            <tab/>
            <ITEXT CH="Protein A affinity"/>
            <tab/>
            <ITEXT CH="1-ml-PAS or similar"/>
            <tab/>
            <ITEXT CH="position 5"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="SEC200Increase_default"/>
            <tab/>
            <ITEXT CH="Gel filtration"/>
            <tab/>
            <ITEXT CH="10/300 Superdex 200 Increase"/>
            <tab/>
            <ITEXT CH="position 1"/>
            <breakline/>
            <tab/>
            <tab/>
            <ITEXT CH="with 0.5ml capillary loop"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="SEC200_16/60_default"/>
            <tab/>
            <ITEXT CH="Gel filtration"/>
            <tab/>
            <ITEXT CH="16/60 Superdex 200"/>
            <tab/>
            <ITEXT CH="position 2"/>
            <breakline/>
            <tab/>
            <tab/>
            <ITEXT CH="with 2 or 2.5ml capillary loop"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="SEC200_26/60_default"/>
            <tab/>
            <ITEXT CH="Gel filtration"/>
            <tab/>
            <ITEXT CH="26/60 Superdex 200"/>
            <tab/>
            <ITEXT CH="position 4"/>
            <breakline/>
            <tab/>
            <tab/>
            <ITEXT CH="with 2.5 or 5ml capillary loop"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="FastDesalting_default"/>
            <tab/>
            <ITEXT CH="Desalting/"/>
            <tab/>
            <ITEXT CH="HR10/10 Sephadex 25"/>
            <tab/>
            <ITEXT CH="position 1"/>
            <breakline/>
            <tab/>
            <ITEXT CH="buffer exchange"/>
            <tab/>
            <ITEXT CH="with 0.5ml capillary loop"/>
            <para PARENT="Table Paragraph"/>
            <ITEXT CH="10. Sample loading into the capillary loop (only for step 2: gel filtration)"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="For the gel filtration and desalting runs, you need to manually load the capillary loop with your sample before you start the run. Depending on your sample volume, you need to choose a volume-matched capillary loop. If you want to maximize your recovery, always use a capillary loop that is at least 30% larger than the volume of your sample. However, if peak separation is your goal, use a very small sample volume, a very small capillary loop and overfill it (e.g. fill a 0.5-ml-loop with 0.8 ml sample and resolve it on the HiLoad 16/600 Superdex pg)."/>
            <para PARENT="Default Paragraph Style"/>
            <ITEXT CH="10.1"/>
            <tab/>
            <ITEXT CH="Attach the correct capillary loop to positions LoopF and LoopE. Wash the loop by connecting a syringe to position Syr and pushing about 10 loop volumes running buffer through the loop."/>
            <para PARENT="List"/>
            <ITEXT CH="10.2 Load your sample into a syringe (e.g. a 1-ml-syringe) avoiding/removing any air bubbles and insert the syringe to position Syr"/>
            <para PARENT="List"/>
            <ITEXT CH="10.3"/>
            <tab/>
            <ITEXT CH="Push the syringe content VERY SLOWLY into the sample loop. Excess sample exits at position W1 and you may recover it."/>
            <para PARENT="List"/>
            <ITEXT CH="11. Starting the method run"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="11.1"/>
            <tab/>
            <ITEXT CH="Click the “folder” in the top left corner of the program window and choose a ready method."/>
            <para PARENT="List"/>
            <ITEXT CH="11.2"/>
            <tab/>
            <ITEXT CH="When the program asks for the column information use the bar code scanner to scan the bar code on the column or find it on the list."/>
            <para PARENT="List"/>
            <ITEXT CH="11.3"/>
            <tab/>
            <ITEXT CH="Double check that all tubes are in correct buffers."/>
            <para PARENT="List"/>
            <ITEXT CH="11.4"/>
            <tab/>
            <ITEXT CH="Start the run by clicking the 'play' button."/>
            <para PARENT="List"/>
            <ITEXT CH="11.5"/>
            <tab/>
            <ITEXT CH="After the run has finnished, you can remove your column. The default methods equilibrate the column in the end to 20% ethanol for storage. Even though it is sold as a single-use device, you can reuse it a couple of times. However, if you use it for a different protein, you need to do rigorous cleaning in between. Deattach the column from its tubings and firmly screw stop plugs to both ends to prevent the column from drying out. Store the column at +4"/>
            <ITEXT FONT="Arial Regular" CH="°"/>
            <ITEXT CH="C. "/>
            <para PARENT="List"/>
            <ITEXT CH="12. Collecting, pooling and concentrating fractions from the affinity chromatography (first step)"/>
            <para PARENT="Heading1"/>
            <ITEXT CH="After the run has finnished, you can look up from the evaluation module, in which fraction(s) the protein eluted and how concentrated it is. You can pool the desired fractions. If your pool has a large volume, you need to concentrate it before you load it to the gel filtration column. Typical gel exclusion columns (Superdex 10/300 GL or Superdex Increase) accept a maximum sample volume of ~0.5 ml (for volumes of ~0.5 to ~1.5 ml, we have the HiLoad 16/600 Superdex pg and for volumes of ~1.5 to ~5 ml, we have the HiLoad 26/600 Superdex pg). Depending on the size of your protein, you should use either the Superdex 75 (proteins from 3 to 70 kDa) or the Superdex 200 (proteins from 10 to 600 kDa). Concentration is done most convieniently by ultrafiltration (e.g Amicon Ultra-4 10K for up to 4 ml, Amicon Ultra-15 10K for up to 15 ml). Because of the high salt and imidazole concentration, it is recommended to dilute the pooled elution fractions 1:1 with water before concentration. Be careful not to overconcentrate (because your protein might precipitate). Overconcentration can happen locally near to the ultrafiltration membrane if you try to concentrate in one centrifugation step. It is better to do several shorter spins and homogenize the concentrate in between to avoid high protein concentrations near the membrane."/>
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