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Gel exclusion chromatography (also called molecular exclusion chromatography, size exclusion chromatography, or gel filtration chromatography) is a low resolution isolation method that employs a cool “trick." This involves the use of beads that have tiny “tunnels" in them that each have a precise size. The size is referred to as an “exclusion limit," which means that molecules above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than the exclusion limit do not enter the tunnels and pass through the column relatively quickly by making their way between the beads. Smaller molecules, which can enter the tunnels, do so, and thus, have a longer path that they take in passing through the column. Because of this, molecules larger than the exclusion limit will leave the column earlier, while those that pass through the beads will elute from the column later. This method allows separation of molecules by their size.
Separation techniques: Chromatography
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification.
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights [1, 2]. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster .
Based on this approach three components form the basis of the chromatography technique.
Stationary phase: This phase is always composed of a “solid” phase or 𠇊 layer of a liquid adsorbed on the surface a solid support”.
Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis gas-liquid chromatography is utilized in the separation of alcohol, esther, lipid, and amino groups, and observation of enzymatic interactions, while molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses .
Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples .
The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achive a satisfactory separation within a suitable timeinterval. Various chromatography methods have been developed to that end. Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, and affinity chromatography .
Types of chromatography
Gel-permeation (molecular sieve) chromatography
Hydrophobic interaction chromatography
High-pressure liquid chromatography (HPLC)
Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of biomolecules. On a column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied ( Figure 1 ). Their flow through inside column material placed on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a tme-, and volume-dependent manner .
Ion- exchange chromatography
Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution . Positively charged ion- exchange matrices are called anion-exchange matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged groups are known as cation-exchange matrices, and adsorb positively charged proteins ( Figure 2 ) .
Ion- exchange chromatography.
Gel- permeation (molecular sieve) chromatography
The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. This procedure is basically used to determine molecular weights of proteins, and to decrease salt concentrations of protein solutions . In a gel- permeation column stationary phase consists of inert molecules with small pores. The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area. Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times ( Figure 3 ) . Sephadeks G type is the most frequently used column material. Besides, dextran, agorose, polyacrylamide are also used as column materials .
Gel-permeation (molecular sieve) chromatography.
This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins . A ligand which can make a complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins leave the column. Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution ( Figure 4 ) .
In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a “liquid-liquid” chromatography .
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used. in column chromatography (alumina, silica gel, cellulose) can be utilized. In this method, the mobile phase travels upward through the stationary phase The solvent travels up the thin plate soaked with the solvent by means of capillary action. During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent .
In cases where molecules of the sample are colorless, florescence, radioactivity or a specific chemical substance can be used to produce a visible coloured reactive product so as to identify their positions on the chromatogram. Formation of a visible colour can be observed under room light or UV light. The position of each molecule in the mixture can be measured by calculating the ratio between the the distances travelled by the molecule and the solvent. This measurement value is called relative mobility, and expressed with a symbol Rf. Rf. value is used for qualitative description of the molecules .
In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N2. Mobile phase which is an inert gas is passed through a column under high pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase. The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes .
Dye- ligand chromatography
Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye . The planar ring structure with negatively charged groups is analogous to the structure of NAD. This analogy has been evidenced by demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is 10-fold stronger rhat that of the affinity of other adsorbents. Under appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange property of adsorbent, the adsorbed proteins are separated from the column [20, 21].
Hydrophobic interaction chromatography (HIC)
In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used. HIC technique is based on hydrophobic interactions between side chains bound to chromatography matrix [22, 23].
Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography (IMAC) .
High-prssure liquid chromatography (HPLC)
Using this chromatography technique it is possible to perform structural, and functional analysis, and purification of many molecules within a short time, This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules, In HPLC, mobile phase passes throuıgh columns under 10 atmospheric pressure, and with a high (0.1𠄵 cm//sec) flow rate. In this technique, use of small particles, and application of high presure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued .
Application areas of chromatography in medicine
Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids, barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and proteins.
Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments. With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method. Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins. Besides, these methods are used to control purity of a protein. HPLC technique which has many superior features including especially its higher sensitivity, rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.
Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
Queen's University Belfast, Medical Biology Centre, Belfast, U.K.
Queen's University Belfast, Medical Biology Centre, Belfast, U.K.
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Size-exclusion or gel filtration chromatography is one of the most popular methods for determining the sizes of proteins. Proteins in solution, or other macromolecules, are applied to a column with a defined support medium. The behavior of the protein depends on its size and that of the pores in the medium. If the protein is small relative to the pore size, it will partition into the medium and emerge from the column after larger proteins. Besides a protein's size, this technique can also be used for protein purification, analysis of purity, and study of interactions between proteins. In this unit protocols are provided for size-exclusion high-performance liquid chromatography (SE-HPLC) and for conventional gel filtration, including calibration of columns (in terms of the Stokes radius) using protein standards.
Membrane-based steric exclusion chromatography for the purification of a recombinant baculovirus and its application for cell therapy
The continuously increasing potential of stem cell treatments for various medical conditions has accelerated the need for fast and efficient purification techniques for individualized cell therapy applications. Genetic stem cell engineering is commonly done with viral vectors like the baculovirus. The baculovirus is a safe and efficient gene transfer tool, that has been used for the expression of recombinant proteins for many years. Its purification has been based mainly on ion exchange matrices. However, these techniques impair process robustness, if different genetically modified virus particles are applied. Here, we evaluated the membrane-based steric exclusion chromatography for the purification of insect cell culture-derived recombinant Autographa californica multicapsid nucleopolehydroviruses for an application in cell therapy. The method has already proven to be a powerful tool for the purification of Influenza A virus particles, using cellulose membranes. Aside from the aforementioned cellulose, we evaluated alternative stationary phases, such as glass fiber and polyamide membranes. The highest dynamic binding capacitiy was determined for cellulose with 5.08E + 07 pfu per cm² membrane. Critical process parameters were optimized, using a design of experiments (DoE) approach. The determined process conditions were verified by different production batches, obtaining a mean virus yield of 91% ± 6.5%. Impurity depletion was >99% and 85% for protein and dsDNA, without nuclease treatment. Due to the method's specificity, its application to other baculoviruses, with varying surface modifications, is conceivable without major process changes. The physiological buffer conditions enable a gentle handling of the virus particles without decreasing the transduction efficacy. The simple procedure with sufficient impurity removal enables the substitution of time-consuming ultra centrifugation steps and can serve as a first process unit operation to obtain higher purities.
Keywords: Baculovirus Design of experiments Downstream processing Stem cells.
Magnetic beads are used to purify recombinant fusion tagged protein using a magnetic rack or platform to separate beads from wash and elution fractions. This procedure allows for rapid processing under native or denaturing conditions within seconds, and is suitable for low throughput using single microfuge tubes or higher throughput using 96-well plates and automation.
BugBuster ® purification kits combine affinity resin, wash buffers, elution buffers, and extraction reagent for convenient preparation of soluble cell extracts and affinity purification of tagged fusion proteins from E. coli.
Antibody purification: ammonium sulfate fractionation or gel filtration
Antibodies can be purified by a variety of methods based on their unique physical and chemical properties such as size, solubility, charge, hydrophobicity and binding affinity. This chapter focuses on ammonium sulfate precipitation as a convenient first step in antibody purification in that, it allows the concentration of the starting material and the precipitation of the desired protein. The principle of ammonium sulfate precipitation lies in "salting out" proteins from the solution. The proteins are prevented to form hydrogen bonds with water and the salt facilitates their interaction with each other forming aggregates that afterward precipitate out of solution. Gel filtration or size- exclusion chromatography is also discussed in this chapter. Gel filtration is based on the relative size of protein molecules and it is of great value to separate IgMs, exchange buffers and/or desalt solutions. The columns designed to separate the proteins are composed of porous beads and the proteins will flow through the packed column inside and around the beads, depending on its size.
Analysis of protein complexes in Arabidopsis leaves using size exclusion chromatography and label-free protein correlation profiling
Protein complexes are fundamentally important for diverse cellular functions, and create functionalities that could never be achieved by a single polypeptide. Knowledge of the protein complex assemblies that exist in plant cells are limited. To close this gap, we applied an integrative proteomic approach that combines cell fractionation, protein chromatography and quantitative mass spectrometry (MS) to analyze the oligomerization state of thousands of proteins in a single experiment. Soluble extracts from intact Arabidopsis leaves were fractionated using size exclusion chromatography (SEC), and abundance profiles across the column fractions were quantified using label-free precursor ion (MS1) intensity. In duplicate experiments, we reproducibly detected 1693 proteins, of which 983 proteins were cytosolic. Based on the SEC profiles, approximately one third of all of the soluble proteins were predicted to be oligomeric. Our dataset includes both subunits of previously known complexes as well as hundreds of new protein complexes. The label-free MS1-based quantification method described here produced a highly useful dataset for the plant biology community, and provided a foundation to incorporate orthogonal protein complex separation methods so the composition and dynamics of protein complexes can be analyzed based on LC/MS profile data alone.
Keywords: Arabidopsis Mass spectrometry Protein complex Proteomics Size exclusion chromatography.
- Desalting &mdash A common use of SEC is for desalting protein or nucleic acid samples. The molecule of interest is eluted in the void volume, while smaller molecules are retained in the gel pores. To obtain the desired separation, the gel should have an exclusion limit significantly smaller than the molecule of interest
- Fractionation &mdash Molecules of varying molecular weights are separated within the gel matrix. With this separation method, the molecules of interest should fall within the fractionation range of the gel
Bio-Rad offers a number of media choices for SEC:
- media for MW separations under 100 KD and for desalting. media for the separation of lipophilic polymers. 1.5m media, process scale, for the purification for antibodies and aggregates.
Size Exclusion Chromatography Media Selection Guide
|Hydrated Bead Size, µm||Hydrated Bed |
|Typical Flow Rate , cm/hr*||Molecular Weight Fractionation Range|
|Bio-Gel P&ndash2, fine||45&ndash90||3 ml/g||5&ndash10||100&ndash1,800|
|Bio-Gel P&ndash2, extra fine||<45||<10|
|Bio-Gel P&ndash4, medium||90&ndash180||4 ml/g||15&ndash20||800&ndash4,000|
|Bio-Gel P&ndash4, fine||45&ndash90||10&ndash15|
|Bio-Gel P&ndash4, extra fine||<45||<10|
|Bio-Gel P&ndash6, medium||90&ndash180||6.5 ml/g||15&ndash20||1,000&ndash6,000|
|Bio-Gel P&ndash6, fine||45&ndash90||10&ndash15|
|Bio-Gel P&ndash6, extra fine||<45||<10|
|Bio-Gel P&ndash6DG gel||90&ndash180||15&ndash20|
|Bio-Gel P&ndash10 gel, medium||90&ndash180||7.5 ml/g||15&ndash20||1,500&ndash20,000|
|Bio-Gel P&ndash10 gel, fine||45&ndash90||10&ndash15|
|Bio-Gel P&ndash30 gel, medium||90&ndash180||9 ml/g||7&ndash13||2,400&ndash40,000|
|Bio-Gel P&ndash30 gel, fine||45&ndash90||6&ndash11|
|Bio-Gel P&ndash60 gel, medium||90&ndash180||11 ml/g||4&ndash6||3,000&ndash60,000|
|Bio-Gel P&ndash60 gel, fine||45&ndash90||3&ndash5|
|Bio-Gel P&ndash100 gel, medium||90&ndash180||12 ml/g||4&ndash6||5,000&ndash100,000|
|Bio-Gel P&ndash100 gel, fine||45&ndash90||3&ndash5|
* Row rates determined in a 1.5 x 70 cm column using a hydrostatic pressure head-to-bed ratio of 1:1.
As there may be limitations to available equipment it should be noted that we have performed this experiment successfully for several years without the use of fraction collectors. One student can easily perform the CMC separation whereas two students are required to perform the Sephadex G-75 separation, one to count drops and collect fractions, the other to maintain the constant buffer head height. If one wanted to extend this experiment, different parameters could be altered to illustrate that resolution is affected by other factors. We believe that the variation of flow rate (0.3–0.7 ml/min) from one group to another is responsible for the greater variance of results for the Sephadex G-75 separation compared with the CMC separation.
The CMC chromatography also lends itself to modifications to illustrate the various aspects of ion exchange chromatography. Instead of eluting Mb with a change in pH to its isoelectric point, one could perform stepwise salt gradients (or a linear salt gradient if one has the equipment) while maintaining the same pH, or one could perform pH gradient elutions.
We are able to perform the separations as described in a 4-h period however students must be ready and organized to accomplish all the tasks involved. We are fortunate that each group has access to a Genesys 2 or 5 UV-visible spectrophotometer thus preventing delays in reading the samples. Having a fraction collector for use in the G-75 separation is extremely helpful as it allows the third member of the group to start adding the water to the fractions (both G-75 and CMC) and to begin reading the absorbances.
This experiment has evolved over a number of years to its current configuration. The present experiment incorporates elements of three previous experiments, a gel filtration experiment with different colored biological polymers (blue dextran, hemoglobin, and cytochrome c) and the dye bromphenol blue, an ion exchange experiment separating the proteins alkaline phosphatase and cytochrome c, and the study of the properties of Mb [ 9 ].
In developing this experiment, the CMC cation exchange column was chosen, because Mb (pI = 7.0 [ 11 ]) at pH 5.6 is positively charged and therefore binds to the column matrix. As the CMC is relatively inexpensive we have not attempted to recycle it after the experiment is completed. Sephadex G-75–120 and G-100–120 (both with 40–120-μm dry bead diameters) were evaluated in the developmental stages of this experiment. Though both have acceptable fractionation ranges, 3–80 kDa for Sephadex G-75 and 4–100 kDa for Sephadex G-100 [ 12 ], we found that the Sephadex G-75 gave an overall better separation with reasonable flow rates for completing the experiment in the allotted time. After the experiment is completed the Sephadex G-75 is thoroughly washed with an aqueous 0.1% (w/v) sodium azide solution (to prevent bacterial growth) and stored at 4 °C until needed.
With respect to safety issues we have not encountered any majors concerns because of the relatively innocuous aqueous conditions of this experiment. The only minor concerns we have noted is adequate ventilation to deal with the stench of the 2-mercatoethanol in the SDS sample dilutor and a warning to the students not to get the Coomassie reagents (Bradford and SDS staining solutions) on their skin.
It was our intention in revising our biochemistry lab curriculum to go toward a group project, cooperative learning setting [ 13 – 16 ] that we believed allows students to gain a clearer understanding of the underpinning of each experiment, in this case the various aspects of protein purification. In addition, we have been concerned over the years about how uncritically students evaluated the information that is collected in the lab. Though we have not formally evaluated student reaction to student collaboration as others have [ 17 ], we believe that overall the collaborative effort is a positive experience. We detail below some of our findings over the last four years.
The three big benefits from group work that we have noticed are as follows: 1) students are more engaged in what they are doing 2) students are more resourceful in terms of managing their time 3) we are able to accomplish much more experimentally in the lab.
There is noticeably more noise in the lab with group work students are talking about what to do and why it is being done instead of silently reading directions and going through the motions. After some rocky starts students make noticeable gains in time management and efficiency. We deliberately create enough tasks such that only by properly coordinating and timing do students finish early, and we ramp up the number of tasks as the semester progresses. Though there are more sets of hands working on the same effort, we have found that those hands are doing more tasks more pipetting, more spectral measurements, more loading of samples. The major role of the instructor is to make certain that each student is involved in all tasks in each experiment. We have not rigorously evaluated whether students learn more or better. We do know that we present students with more to learn.
On the negative side, a major problem every semester is student responsibility vis a vis his/her group. There is on occasion significant resentment of the student who does not pull his/her weight in the group, especially in terms of working on joint reports. Also the student(s) doing most of the work believe that his/her/their larger contribution is not recognized by the instructor. On the flip side the standard student excuse for not being involved in the writing is not being able to make connections with one's group. The quality of group reports is thus very spotty and “committee” efforts are sometimes disastrous with one report section having no connection to another. We have attempted different models for constructing groups but have not come up with one that is noticeably better than another each have their unique deficiencies. We do have a self- and peer evaluation at the end of the semester that is coupled with an instructor evaluation (10% of total lab grade) that has had some effect in forcing students to be more conscientious in their group work. Additionally, the self- and peer evaluations allow the students to gain some experience that will be useful when they are in a supervisory position later in their careers.