Name: protein purification with sec and imac
Uploaded: 2023-04-30T06:30:54.000Z
Duration: 7 min 51 s
Channel: JoVE

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High School

Life Science

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Matter and Energy in Organisms and Ecosystems

HS-LS1-6.

chromatography-based-biomolecule-purification-methods

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.

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>

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

"Chromatography" refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule'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.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 "exclusion limit" 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 "permeation limit".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've seen the theory of SEC, let's look at how it is carried out.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.Now let'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 "normal-" or "reverse-role". 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'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 "tag": short peptide sequences engineered to bind to the ligand.One variety is immobilized metal ion affinity chromatography, "IMAC" 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.?Now that you've seen the theory of affinity chromatography, let's look at an IMAC procedure in the laboratory.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.Now that we've seen the theory and procedure of these two techniques, let's look at some of the ways they'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'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.You've just watched JoVE'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!

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Biochemistry

protein purification with sec and imac

Protein purification with size-exclusion chromatography and affinity chromatography helps isolate biomolecules from complex mixtures. These methods are common in biochemistry because purified samples are needed before a biomolecule’s properties and activity can be studied. The video focuses on the principles behind size-exclusion chromatography, affinity chromatography, and immobilized metal affinity chromatography, or IMAC.

Size-exclusion chromatography, or SEC, separates molecules by size. A sample moves through a column packed with porous beads, which make up the stationary phase. Large molecules that cannot enter the pores travel the shortest path and leave first. Smaller molecules enter the pores, spend more time in the column, and leave later. Molecules between the exclusion limit and the permeation limit can be separated from each other, while molecules above or below those limits exit together.

The SEC procedure begins with equilibration of the column using deionized water and chromatography buffer. Sample-containing buffer is then injected, and the liquid is pushed through at a low flow rate. A detector watches what comes out of the column, and fractions are collected in tubes. These fractions can then be checked for the target molecule by gel electrophoresis or other analytical methods.

Affinity chromatography, or AC, uses the fact that many biomolecules bind selectively to certain ligands. In this method, a target-specific ligand is attached to the stationary phase. As the mixture passes through the column, the target binds to the ligand while the rest flows through. The target can then be released by biospecific elution, which may use a competing agent in normal-role or reverse-role form, or by nonspecific elution, which changes pH, ionic strength, or polarity.

If a protein does not naturally bind to an immobilized ligand, it can be engineered with a tag. A common example is a polyhistidine tag, or his-tag. In immobilized metal ion affinity chromatography, nickel or cobalt on the column binds to histidine residues through the imidazole side chain. The tagged protein is then released with free imidazole by reverse-role specific elution and can be used in downstream applications.

The IMAC procedure begins by mixing the stationary phase with the sample so the his-tagged protein can bind. The slurry is poured into the column, and unbound material drains to waste. The resin is rinsed into the column, stirred, and washed to remove leftover unbound components. After that, a container is placed to collect the target protein, and imidazole buffer is added to release it. The imidazole binds the metal, displaces the tagged protein, and the collected step may be repeated to improve recovery. For further cleanup, SEC can be run before analysis.

These purification methods are useful in biochemical and biomedical research. One example is cystic fibrosis research, where the cystic fibrosis transmembrane conductance regulator protein, or CFTR, is grown with a his-tag in yeast and then isolated with affinity and size-exclusion chromatography. In some cases, a polyhistidine tag can change protein structure and affect function. Another tag used in protein work is maltose-binding protein, or MBP, which binds amylose in a column and is released with maltose. The MBP can then be cleaved and removed with SEC to produce the desired pure protein.

Protein Purification with SEC and IMAC

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Protein purification with size-exclusion chromatography and affinity chromatography helps isolate biomolecules from complex mixtures. These methods are common in biochemistry because purified samples are needed before a biomolecule’s properties and activity can be studied. The video focuses on the principles behind size-exclusion chromatography, affinity chromatography, and immobilized metal affinity chromatography, or IMAC. Size-exclusion chromatography, or SEC, separates molecules by size. A...

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biochemistry

This collection presents commonly used purification methods, such as affinity chromatography, as well as analytical methods, like MALDI-TOF. In addition, the videos showcase methods for assessing biomolecule interaction and function, such as co-immunoprecipitation and metabolic labeling.

Explore how size-exclusion chromatography and IMAC separate proteins by size or binding, and see how tagged proteins are purified for research.

Protein Purification with SEC and IMAC | JoVE K12

Overview

Size-Exclusion Chromatography

Operation of Size-Exclusion Chromatography

Affinity Chromatography

Operation of Affinity Chromatography

Applications

Summary

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Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

Column Chromatography

Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This ...

bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

Construct and revise an explanation based on evidence for how carbon, hydrogen, and oxygen from sugar molecules may combine with other elements such as nitrogen, sulfur, and phosphorus to form amino acids and other carbon-based molecules.

Protein Purification with SEC and IMAC

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