This work was funded by National Institutes of Health grant GM086822 and National Science Foundation major research instrumentation grant DBI-0960313. The authors would like to thank Drs. Jon Miyake &amp; Donna Hardy (Bio-Rad) and Jennifer Loertscher (Seattle University) for their technical expertise. Select chromatography reagents and supplemental instrumentation were generously provided by Bio-Rad.

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Select chromatography reagents and supplemental instrumentation were provided by Bio-Rad.

Liquid chromatography techniques have proven invaluable for preparing highly purified proteins necessary for conducting immunological 6, biochemical 7, and structural 8 studies. HIC purification methods most often require empirical determination of a preferred medium, and ligand structure, ligand density, and matrix bead properties all have been shown to impact chromatographic results 2, 3. Automated column scouting is an efficient approach for selecting a HIC medium for subsequent optimization and protein purification 4. The automated column scouting method presented can be readily adapted to various HIC protein purification strategies. Alterations in salt concentration, choice of salt, salt gradient, and pH may further improve purification conditions, and the effects of varying these essential parameters have been previously reviewed 13,14. HIC eluate containing a partially purified target protein can be further purified using a complementary chromatographic technique, such as ion exchange chromatography (IEX) or gel filtration/size exclusion chromatography 9,10.
Because of its unique light absorbance and emission characteristics, the elution profile of GFP can be identified using in-line and post-run approaches. To that end, this protocol can further be adapted for purification of recombinant GFP-fusion proteins, in which an unrelated target protein is &#x201C;tagged&#x201D; with GFP. GFP-tagged target proteins can be detected with UV light 11 and purified using various chromatographic approaches, including the HIC purification strategy described above. GFP purification has also become a pedagogical staple in biochemistry labs for the teaching of modern protein science techniques 12.
While basic HIC protein purification can be conducted using a substantially less robust chromatography system, the instrumentation presented here has a number of distinct advantages to aid in obtaining favorable results. Notable benefits of utilizing a highly automated system include improved run-to-run condition reproducibility, time-savings, and decreased opportunities for air to be introduced into the system 10. System-controlled buffer blending, which is facilitated by the system maximizer, allows for increased consistency in buffer preparation and experimental reproducibility. Drawing enough sample load into the dynamic sample loop for all scouting runs further ensures uniformity of the sample being added to each column and allows for sequential runs without interruption or manual reloading. Controlled sample injection from the sample loop onto the column decreases variability that may occur with manual sample loading. A pair of column selection valves allow for consecutive sample runs, each using a different HIC column, without having to replumb the system. Performing multi-wavelength analysis is particularly beneficial when assaying a protein with a unique spectrophotometric profile. In addition to GFP, cytochromes, flavoproteins, and other heme-containing proteins may benefit from this technique. Conductivity and pH monitoring devices allow for verification of real-time experimental conditions. Split fraction eluate collection allows for improved fraction handling and enables easy transfer for post-run analysis methods that require a minimal sample volume (e.g. ELISA, SDS-PAGE, western blot, and Experion microfluidic electrophoresis). While operating the second fraction collector offline requires manual synchronization, it allows for maximum flexibility in fraction collector selection. The most significant drawbacks to utilizing such a robust chromatography system for HIC column scouting and protein purification include the initial time and budgetary expenditures associated with instrument acquisition and operator training. 
The protocol presented here utilizes a Bio-Rad DuoFlow chromatography system; however, equally robust instrumentation from other manufacturers, such as the &Auml;KTA Avant from GE Healthcare, may also be utilized and are capable of producing equivalent results. Even comparable chromatography system have unique characteristics (e.g. method programming, component nomenclature, operator preference, and scalability limitations) that should be considered before initiating a purification procedure or instrument acquisition.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td><a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">BioLogic DuoFlow Pathfinder 20 System</a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257 </a></td>
 <td>Includes system maximizer, mixer, F10 workstation, AVR7-3 valve, QuadTec UV/Vis detector with flow cell, BioFrac fraction collector, BioLogic software, and starter kit</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8 stream-select valve </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408 </a></td>
 <td>High-pressure valve, 9-port, 8-position, 3,500 psi (233 bar) limit, for use with BioLogic DuoFlow system</td>
 </tr>
 <tr>
 <td>Diverter valve SV3T-2 </td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td>7600410 </td>
 <td>Solenoid valve, 3-port, 2-position, 30 psi (2 bar) limit, for use with BioLogic DuoFlow system</td>
 </tr>
 <tr>
 <td>Stream splitter valve</td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">DynaLoop 25 Kit </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451 </a></td>
 <td>Sliding-piston sample loop kit, includes 25 ml DynaLoop sliding piston loop, DynaLoop parts kit (#750-0450) </td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFrac Fraction Collector</a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a></td>
 <td>Two fraction collectors used</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFrac microplate drop head adapter</a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFrac microtiter plate adapter</a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">7410017</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">UV Optics Module </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202 </a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">Halogen lamp </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">7601331 </a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">Econo Gradient Pump </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001 </a></td>
 <td>gradient pump for low-pressure protein purification, includes tubing and fittings kit </td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion System </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">7007001 </a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260 Analysis Kit </a></td>
 <td><a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a></td>
 <td><a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102 </a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HiTrap HIC column selection kit</td>
 <td>GE Healthcare</td>
 <td>28-4110-07 </td>
 <td>Includes 7 x 1 ml pre-packed columns of HIC media for scouting</td>
 </tr>
 <tr>
 <td>GE Healthcare-to-Bio-Rad column fittings</td>
 <td>GE Healthcare</td>
 <td>18111251 and 18111257</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

automated hydrophobic interaction chromatography column selection for use in protein purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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43.1K Views.  Seattle University. An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.