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.

<ol>
	<li>Cummins, P. M., O'Connor, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography.">Hydrophobic interaction chromatography.</a> Methods Mol. Biol. 681, 431-437 (2011).</li><li>Nagrath, D., Xia, F., Cramer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+modeling+of+nonlinear+hydrophobic+interaction+chromatographic+systems.">Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems.</a> J. Chromatogr. A. 1218, 1219-1226 (2011).</li><li>To, B. C., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.+IV.+Protein+adsorption+capacity+and+transport+in+preparative+mode.">Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.</a> J. Chromatogr. A. 1218, 427-440 (2011).</li><li>Huddleston, J. G., Wang, R., Lyddiatt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+mild+hydrophobic+interaction+chromatography+for+"method+scouting"+protein+purification+strategies+in+aqueous+two-phase+systems:+a+study+using+model+proteins.">On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins.</a> Biotechnol Bioeng. 44, 626-635 (1994).</li><li>Arun, K. H., Kaul, C. L., Ramarao, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+proteins+in+receptor+research:+an+emerging+tool+for+drug+discovery.">Green fluorescent proteins in receptor research: an emerging tool for drug discovery.</a> J. Pharmacol. Toxicol. Methods. 51, 1-23 (2005).</li><li>Terrizzi, S. C., Banh, C., Brossay, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.A+Protocol+for+the+Production+of+KLRG1+Tetramer.">L.A Protocol for the Production of KLRG1 Tetramer.</a> J. Vis. Exp. (35), e1701-e1701 (2010).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+Magnetosome+Associated+Protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 Magnetosome Associated Protein MamA&#916;41.</a> J. Vis. Exp. (37), 10-3791 (2010).</li><li>Kong, Y., Li, X., Bai, G., Ma, G., Su, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+system+for+multidimensional+integrated+protein+chromatography.">An automatic system for multidimensional integrated protein chromatography.</a> J. Chromatogr. A. 1217, 6898-6904 (2010).</li><li>Camper, D. V., Viola, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+protein+purification.">Fully automated protein purification.</a> Anal. Biochem. 393, 176-181 (2009).</li><li>Hammon, J., Palanivelu, D. V., Chen, J., Patel, C., Minor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+green+fluorescent+protein+screen+for+identification+of+well-expressed+membrane+proteins+from+a+cohort+of+extremophilic+organisms.">A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.</a> Protein Sci. 18, 121-133 (2009).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochem. Mol. Biol. Educ. 36, 43-54 (2008).</li><li>Queiroz, J. A., Tomaz, C. T., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.">Hydrophobic interaction chromatography of proteins.</a> J. Biotechnol. 87, 143-159 (2001).</li><li>McCue, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+use+of+hydrophobic+interaction+chromatography+in+protein+purification+applications.">Theory and use of hydrophobic interaction chromatography in protein purification applications.</a> Methods Enzymol. 463, 405-414 (2009).</li></ol>

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><tbody><tr><td>BioLogic DuoFlow Pathfinder 20 System </td><td>Bio-Rad</td><td>7602257</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>AVR9-8 stream-select valve </td><td>Bio-Rad</td><td>7600408</td><td></td></tr><tr><td>Diverter valve SV3-2 </td><td>Bio-Rad</td><td>7500410</td><td></td></tr><tr><td>Stream splitter valve</td><td>Bio-Rad</td><td>7410017</td><td></td></tr><tr><td>DynaLoop 25 Kit </td><td>Bio-Rad</td><td>7500451</td><td></td></tr><tr><td>BioFrac Fraction Collector</td><td>Bio-Rad</td><td>7410002</td><td>Two fraction collectors used</td></tr><tr><td>BioFrac microplate drop head adapter</td><td>Bio-Rad</td><td>74010088</td><td></td></tr><tr><td>BioFrac microtiter plate adapter</td><td>Bio-Rad</td><td>7410017</td><td></td></tr><tr><td>UV Optics Module </td><td>Bio-Rad</td><td>7500202</td><td></td></tr><tr><td>Halogen lamp </td><td>Bio-Rad</td><td>7601331</td><td></td></tr><tr><td>Econo Gradient Pump </td><td>Bio-Rad</td><td>7319001</td><td></td></tr><tr><td>Experion System </td><td>Bio-Rad</td><td>7007001</td><td></td></tr><tr><td>Experion Pro260 Analysis Kit </td><td>Bio-Rad</td><td>7007102</td><td></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></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 ...

automated-hydrophobic-interaction-chromatography-column-selection-for

Research

JoVE Journal

Biology

43.6K Views.  Seattle University. Een automatische methode om geschikte hydrofobe interactie chromatografie (HIC) media te gebruiken in het proces van eiwitzuivering gepresenteerd. De werkwijze maakt gebruik van een medium-druk vloeistof chromatografie systeem met automatische buffer mengen dynamische monsterlus injectie opeenvolgende kolomselektiemiddelen, multi-golflengte analyse en split fractie eluaat collectie.

Video: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Biochemistry

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
</div>
<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
</div>
<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
</div>
<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

</div>
<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
</div>
<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
</div>
<div class="chapter_text" id="script_6">
You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Size Exclusion and Affinity Chromatography

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

Education

JoVE Science Education

Chemistry

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Immunology

Immunotherapy

Immunogenics and Vaccine Development

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Antibody-Based Technologies

Production and Purification

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads

First, open the software to control the mobile phases. Wash UPLC instruments with 50% solvent B and 50% solvent C at the flow rate of 0.2 milliliters per minute, for 30 minutes. Then wash, with 25% solvent A and 75% solvent B at a flow rate of 0.2 milliliters per minute, for 20 minutes, then, at a flow rate of 0.4 milliliters per minute.
Dissolve the labeled N-glycans with 25 microliters of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 (volume/volume) ratio. Then, centrifuge at 134 x g for 5 minutes at 4 degrees Celsius, and use a drive pipe to load 10 microliters of the supernatant into the UPLC instruments.
Separate the labeled N-glycans at a flow rate of 0.4 milliliters per minute, with a linear gradient of 75% to 62% acetonitrile for 25 minutes. Then, perform an analytical run by Dextran Calibration Ladder/Glycopeptide column on a UPLC at 60 degrees Celsius.
Detect N-glycan fluorescence at excitation and emission wavelengths of 330 nanometers and 420 nanometers respectively.

EoE Sub Articles

Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the &#39;wash unbound sample block&#39; when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Samenvatting

Meer video's verkennen