Saturday, January 27, 2018
Size-exclusion chromatography
Size-exclusion chromatography (SEC), also known
as molecular sieve chromatography, is
a chromatographic method in which molecules in solution are separated
by their size, and in some cases molecular weight. It is usually applied
to large molecules or macromolecular complexes such as proteins and
industrial polymers. Typically, when an aqueous solution is used to transport
the sample through the column, the technique is known as gel-filtration
chromatography, versus the name gel permeation chromatography, which is
used when an organic solvent is used as a mobile phase. SEC is a widely
used polymer characterization method because of its ability to
provide good molar mass distribution (Mw) results for polymers.
Applications
The main application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.
Advantages
The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate,[3] and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to separate. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase.
The other advantage to this experimental method is that in certain cases, it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determine how the compound interacts with the gel (stationary phase). To determine approximate molecular weight, the elution volumes of compounds with their corresponding molecular weights are obtained and then a plot of “Kav” vs “log(Mw)” is made, where Kav = (Ve-Vo)/(Vt-Vo) and Mw is the molecular mass. This plot acts as a calibration curve, which is used to approximate the desired compound’s molecular weight. The Ve component represents the volume at which the intermediate molecules elute such as molecules that have partial access to the beads of the column. In addition, Vt is the sum of the total volume between the beads and the volume within the beads. The Vo component represents the volume at which the larger molecules elute, which elute in the beginning. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there must be a 10% difference in molecular mass to have a good resolution.
Discovery
The technique was invented by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte’s Hospital,London
There were also attempts to fractionate synthetic high polymers; however, it was not until 1964, when J. C. Moore of the Dow Chemical Company published his work on the preparation of gel permeation chromatography (GPC) columns based on cross-linked polystyrene with controlled pore size, that a rapid increase of research activity in this field began. It was recognized almost immediately that with proper calibration, GPC was capable to provide molar mass and molar mass distribution information for synthetic polymers. Because the latter information was difficult to obtain by other methods, GPC came rapidly into extensive use.
Theory and method
SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the adsorbent materials adsorption ("stationary phases"). This process is usually performed with a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time.
One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule elutes late (after the molecule has penetrated all of the pore- and interparticle volume—approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (~35% of the column volume) and elutes earlier when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.
However, as there are various measures of the size of a macromolecule (for instance, the radius of gyration and the hydrodynamic radius), a fundamental problem in the theory of SEC has been the choice of a proper molecular size parameter by which molecules of different kinds are separated. Experimentally, Benoit and co-workers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume, for several different chain architecture and chemical compositions. The observed correlation based on the hydrodynamic volume became accepted as the basis of universal SEC calibration.
Still, the use of the hydrodynamic volume, a size based on dynamical properties, in the interpretation of SEC data is not fully understood. This is because SEC is typically run under low flow rate conditions where hydrodynamic factor should have little effect on the separation. In fact, both theory and computer simulations assume a thermodynamic separation principle: the separation process is determined by the equilibrium distribution (partitioning) of solute macromolecules between two phases --- a dilute bulk solution phase located at the interstitial space and confined solution phases within the pores of column packing material. Based on this theory, it has been shown that the relevant size parameter to the partitioning of polymers in pores is the mean span dimension (mean maximal projection onto a line). Although this issue has not been fully resolved, it is likely that the mean span dimension and the hydrodynamic volume are strongly correlated.
Factors affecting filtration
In real-life situations, particles in solution do not have a fixed size, resulting in the probability that a particle that would otherwise be hampered by a pore passing right by it. Also, the stationary-phase particles are not ideally defined; both particles and pores may vary in size. Elution curves, therefore, resemble Gaussian distributions. The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue.
Like other forms of chromatography, increasing the column length enhances resolution, and increasing the column diameter increases column capacity. Proper column packing is important for maximum resolution: An over-packed column can collapse the pores in the beads, resulting in a loss of resolution. An under-packed column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution.
Analysis
In simple manual columns, the eluent is collected in constant volumes, known as fractions. The more similar the particles are in size the more likely they are in the same fraction and not detected separately. More advanced columns overcome this problem by constantly monitoring the eluent.
The collected fractions are often examined by spectroscopic techniques to determine the concentration of the particles eluted. Common spectroscopy detection techniques are refractive index (RI) and ultraviolet (UV). When eluting spectroscopically similar species (such as during biological purification), other techniques may be necessary to identify the contents of each fraction. It is also possible to analyse the eluent flow continuously with RI, LALLS, Multi-Angle Laser Light Scattering MALS, UV, and/or viscosity measurements.
The elution volume (Ve) decreases roughly linear with the logarithm of the molecular hydrodynamic volume. Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight), and a sample containing a very large molecule such as thyroglobulin to determine the void volume. (Blue dextran is not recommended for Vo determination because it is heterogeneous and may give variable results) The elution volumes of the standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and plotted against the log of the standards' molecular weights.
Applications
Biochemical applications
In general, SEC is considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final step of a purification. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. SEC can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form elutes much later due to its smaller size.
Polymer synthesis
SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer, that is, the ability to find the distribution of the sizes of polymer molecules. If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis (often THF). In alternative fashion, techniques such as light scattering and/or viscometry can be used online with SEC to yield absolute molecular weights that do not rely on calibration with standards of known molecular weight. Due to the difference in size of two polymers with identical molecular weights, the absolute determination methods are, in general, more desirable. A typical SEC system can quickly (in about half an hour) give polymer chemists information on the size and polydispersity of the sample. The preparative SEC can be used for polymer fractionation on an analytical scale.
Drawback
In SEC, mass is not measured so much as the hydrodynamic volume of the polymer molecules, that is, how much space a particular polymer molecule takes up when it is in solution. However, the approximate molecular weight can be calculated from SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only an approximate measurement can be obtained. Another drawback is the possibility of interaction between the stationary phase and the analyte. Any interaction leads to a later elution time and thus mimics a smaller analyte size.
When performing this method, the bands of the eluting molecules may be broadened. This can occur by turbulence caused by the flow of the mobile phase molecules passing through the molecules of the stationary phase. In addition, molecular thermal diffusion and friction between the molecules of the glass walls and the molecules of the eluent contribute to the broadening of the bands. Besides broadening, the bands also overlap with each other. As a result, the eluent usually gets considerably diluted. A few precautions can be taken to prevent the likelihood of the bands broadening. For instance, one can apply the sample in a narrow, highly concentrated band on the top of the column. The more concentrated the eluent is, the more efficient the procedure would be. However, it is not always possible to concentrate the eluent, which can be considered as one more disadvantage.
Applications
The main application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.
Advantages
The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate,[3] and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to separate. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase.
The other advantage to this experimental method is that in certain cases, it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determine how the compound interacts with the gel (stationary phase). To determine approximate molecular weight, the elution volumes of compounds with their corresponding molecular weights are obtained and then a plot of “Kav” vs “log(Mw)” is made, where Kav = (Ve-Vo)/(Vt-Vo) and Mw is the molecular mass. This plot acts as a calibration curve, which is used to approximate the desired compound’s molecular weight. The Ve component represents the volume at which the intermediate molecules elute such as molecules that have partial access to the beads of the column. In addition, Vt is the sum of the total volume between the beads and the volume within the beads. The Vo component represents the volume at which the larger molecules elute, which elute in the beginning. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there must be a 10% difference in molecular mass to have a good resolution.
Discovery
The technique was invented by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte’s Hospital,
There were also attempts to fractionate synthetic high polymers; however, it was not until 1964, when J. C. Moore of the Dow Chemical Company published his work on the preparation of gel permeation chromatography (GPC) columns based on cross-linked polystyrene with controlled pore size, that a rapid increase of research activity in this field began. It was recognized almost immediately that with proper calibration, GPC was capable to provide molar mass and molar mass distribution information for synthetic polymers. Because the latter information was difficult to obtain by other methods, GPC came rapidly into extensive use.
Theory and method
SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the adsorbent materials adsorption ("stationary phases"). This process is usually performed with a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time.
One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule elutes late (after the molecule has penetrated all of the pore- and interparticle volume—approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (~35% of the column volume) and elutes earlier when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.
However, as there are various measures of the size of a macromolecule (for instance, the radius of gyration and the hydrodynamic radius), a fundamental problem in the theory of SEC has been the choice of a proper molecular size parameter by which molecules of different kinds are separated. Experimentally, Benoit and co-workers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume, for several different chain architecture and chemical compositions. The observed correlation based on the hydrodynamic volume became accepted as the basis of universal SEC calibration.
Still, the use of the hydrodynamic volume, a size based on dynamical properties, in the interpretation of SEC data is not fully understood. This is because SEC is typically run under low flow rate conditions where hydrodynamic factor should have little effect on the separation. In fact, both theory and computer simulations assume a thermodynamic separation principle: the separation process is determined by the equilibrium distribution (partitioning) of solute macromolecules between two phases --- a dilute bulk solution phase located at the interstitial space and confined solution phases within the pores of column packing material. Based on this theory, it has been shown that the relevant size parameter to the partitioning of polymers in pores is the mean span dimension (mean maximal projection onto a line). Although this issue has not been fully resolved, it is likely that the mean span dimension and the hydrodynamic volume are strongly correlated.
Factors affecting filtration
In real-life situations, particles in solution do not have a fixed size, resulting in the probability that a particle that would otherwise be hampered by a pore passing right by it. Also, the stationary-phase particles are not ideally defined; both particles and pores may vary in size. Elution curves, therefore, resemble Gaussian distributions. The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue.
Like other forms of chromatography, increasing the column length enhances resolution, and increasing the column diameter increases column capacity. Proper column packing is important for maximum resolution: An over-packed column can collapse the pores in the beads, resulting in a loss of resolution. An under-packed column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution.
Analysis
In simple manual columns, the eluent is collected in constant volumes, known as fractions. The more similar the particles are in size the more likely they are in the same fraction and not detected separately. More advanced columns overcome this problem by constantly monitoring the eluent.
The collected fractions are often examined by spectroscopic techniques to determine the concentration of the particles eluted. Common spectroscopy detection techniques are refractive index (RI) and ultraviolet (UV). When eluting spectroscopically similar species (such as during biological purification), other techniques may be necessary to identify the contents of each fraction. It is also possible to analyse the eluent flow continuously with RI, LALLS, Multi-Angle Laser Light Scattering MALS, UV, and/or viscosity measurements.
The elution volume (Ve) decreases roughly linear with the logarithm of the molecular hydrodynamic volume. Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight), and a sample containing a very large molecule such as thyroglobulin to determine the void volume. (Blue dextran is not recommended for Vo determination because it is heterogeneous and may give variable results) The elution volumes of the standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and plotted against the log of the standards' molecular weights.
Applications
Biochemical applications
In general, SEC is considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final step of a purification. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. SEC can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 Å and 36 Å for the folded and unfolded forms, respectively. SEC allows the separation of these two forms, as the folded form elutes much later due to its smaller size.
Polymer synthesis
SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer, that is, the ability to find the distribution of the sizes of polymer molecules. If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis (often THF). In alternative fashion, techniques such as light scattering and/or viscometry can be used online with SEC to yield absolute molecular weights that do not rely on calibration with standards of known molecular weight. Due to the difference in size of two polymers with identical molecular weights, the absolute determination methods are, in general, more desirable. A typical SEC system can quickly (in about half an hour) give polymer chemists information on the size and polydispersity of the sample. The preparative SEC can be used for polymer fractionation on an analytical scale.
Drawback
In SEC, mass is not measured so much as the hydrodynamic volume of the polymer molecules, that is, how much space a particular polymer molecule takes up when it is in solution. However, the approximate molecular weight can be calculated from SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only an approximate measurement can be obtained. Another drawback is the possibility of interaction between the stationary phase and the analyte. Any interaction leads to a later elution time and thus mimics a smaller analyte size.
When performing this method, the bands of the eluting molecules may be broadened. This can occur by turbulence caused by the flow of the mobile phase molecules passing through the molecules of the stationary phase. In addition, molecular thermal diffusion and friction between the molecules of the glass walls and the molecules of the eluent contribute to the broadening of the bands. Besides broadening, the bands also overlap with each other. As a result, the eluent usually gets considerably diluted. A few precautions can be taken to prevent the likelihood of the bands broadening. For instance, one can apply the sample in a narrow, highly concentrated band on the top of the column. The more concentrated the eluent is, the more efficient the procedure would be. However, it is not always possible to concentrate the eluent, which can be considered as one more disadvantage.
Gel Filtration Chromatography
Gel Filtration Chromatography
Applications
Gel filtration chromatography, a
type of size exclusion chromatography, can be used to either fractionate
molecules and complexes in a sample into fractions with a particular size range,
to remove all molecules larger than a particular size from the sample, or a
combination of both operations. Gel filtration chromatography can be used to
separate compounds such as small molecules, proteins, protein complexes,
polysaccharides, and nucleic acids when in aqueous solution. When an organic
solvent is used as the mobile phase, the process is instead referred to as gel
permeation chromatography.
Gel filtration chromatography can
also be used for:
1. Fractionation
of molecules and complexes within a predetermined size range
2. Size
analysis and determination
3. Removal
of large proteins and complexes
4. Buffer
exchange
5. Desalting
6. Removal
of small molecules such as nucleotides, primers, dyes, and contaminants
7. Assessment
of sample purity
8. Separation
of bound from unbound radioisotopes
Gel filtration chromatography media
for all of the above uses are available in prepacked gravity flow columns, spin
columns, low-pressure and medium-pressure chromatography columns, and bottled
resins.
Gel Filtration Chromatography
Mechanism
In a gel filtration chromatography
column, the stationary phase is composed of a porous matrix, and the mobile
phase is the buffer that flows in between the matrix beads. The beads have a
defined pore size range, known as the fractionation range. Molecules and
complexes that are too large to enter the pores stay in the mobile phase and
move through the column with the flow of the buffer. Smaller molecules and
complexes that are able to move into the pores enter the stationary phase and
move through the gel filtration column by a longer path through pores of the
beads.
Any molecule or complex that is
above the fractionation range for a particular gel filtration chromatography
column will move through the column faster than any molecule that can enter the
stationary phase. Therefore, any constituent in the sample that is above the
fractionation range will elute first (in the void volume) before anything that
is in the fractionation range. The minimum size that will remain in the mobile
phase and not enter the stationary phase is known as the exclusion limit.
Bio-Rad offers gel filtration chromatography media and columns with exclusion
limits ranging over three orders of magnitude, from 100 daltons to 100,000
daltons (100 kDa).
Molecules and complexes that can
enter the stationary phase will be fractionated according to their sizes.
Smaller molecules will migrate deep into the pores and will be retarded more
than larger molecules that do not so easily enter the pores, and are thus
eluted from the column more quickly. This difference in pore migration leads to
fractionation of components by size with the largest eluting first.
In gel filtration chromatography
columns designed for desalting, buffer exchange, and the removal of small
molecules such as nucleotides, the salts and small compounds readily enter the
pores, are retarded, and migrate more slowly through the column than the larger
proteins or nucleic acids. Therefore, the components of interest in the sample
are eluted in advance of salts, nucleotides, etc. DNA cleanup kits using this
mechanism often contain gel filtration spin columns.
Resolution, here defined as the
sharpness of the boundaries between size fractions, is determined by bead size
and a number of other factors. Smaller bead size generally yields higher
resolution in a gel filtration chromatography column. Compact molecules diffuse
through the stationary phase faster than linear molecules. Size exclusion,
fractionation range, and elution rate are affected by buffer composition, ionic
strength, and pH. For the fractionation of complex mixtures of proteins,
elution times and size exclusion limits may need to be determined empirically.
Gel Filtration Chromatography Media
An important criterion for gel
filtration chromatography media is that media is inert and that nothing in the
sample or any buffer binds to the media. Another consideration is the type of
gel filtration column being used and whether it is used in a pressurized
chromatography system or gravity flow or spin columns. If a pressurized
chromatography system is being used, both the column and the media must be able
to tolerate the pressure and flow rates used.
Commonly used media for gel
filtration chromatography are based on agarose or polyacrylamide beads,
dextrose for gravity or low-pressure systems, and polymeric resins for
medium-pressure systems. The choice of media depends on the properties of the
components to be separated and other experimental factors. The following are
general considerations when determining the choice of gel filtration
chromatography media:
l
Fractionation range
l
Size exclusion limit
l
Operating pressure
l
Flow rate
l
Sample viscosity
l
pH range
l
Autoclavability
Tolerance for water-miscible organic
solvents; some samples may be more soluble in a water-organic mix
Tolerance for detergents, chaotropic
agents, formamide, etc.
Operating temperature
The types of samples, choice of
media, and the chromatography system setup will determine which parameters are
the most important for a given purification application.
this symbol highlights
troubleshooting advice to help analyse and resolve .... or for other
chromatography techniques and assays. Gel filtration in group ...
kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdf
kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdf
Gel filtration chromatography
seprarates proteins, prptides, and oligonucleotides on the basis of size.
Molecules move through a bed of porous beads, ...
www.sigmaaldrich.com/.../Protein_Analysis/Chromatography/Gel_Filtration_Chromatography.html
www.sigmaaldrich.com/.../Protein_Analysis/Chromatography/Gel_Filtration_Chromatography.html
Gel filtration chromotography is
based off of the principal of gravity. Most simply, it is a large, vertical
tube. Inside this tube, many tiny beads of ...
student.biology.arizona.edu/honors2003/group04/gelfiltration.html
student.biology.arizona.edu/honors2003/group04/gelfiltration.html
Gel filtration chromatography
separates substances on the basis of their size. ... For gel filtration
chromatography, it is important to apply the sample to ...
www.science.smith.edu/departments/Biochem/Biochem_353/cytoprep.html
www.science.smith.edu/departments/Biochem/Biochem_353/cytoprep.html
Gel filtration chromatography or
perhaps just gel filtration is used to separate or purify protein based on the
size properties. ...
envirodiary.com/2007/05/18/gel-filtration-chromatography-procedures
envirodiary.com/2007/05/18/gel-filtration-chromatography-procedures
Gel
filtration chromatography ge healthcare life sciences
The principle of Gel Filtration Chromatography Separation in Gel Filtration Chromatography is based on the differences in sizes from biomolecules as they ...
www5.gelifesciences.com/.../Content/44B2ACD2F1401137C12572FA00812CF3/
The principle of Gel Filtration Chromatography Separation in Gel Filtration Chromatography is based on the differences in sizes from biomolecules as they ...
www5.gelifesciences.com/.../Content/44B2ACD2F1401137C12572FA00812CF3/
Students use gel-filtration
chromatography to separate three proteins whose molecular weights differ
significantly and whose activities are simple to ...
faculty.mansfield.edu/bganong/biochemistry/gfc3.htm
faculty.mansfield.edu/bganong/biochemistry/gfc3.htm
GEL FILTRATION CHROMATOGRAPHY
(9/16/03 and 9/23/03). Packing the column. ***Note: Do not let the column run
dry. Always have buffer above the level of the ...
www.shsu.edu/~chm_mfp/gel_filtration_chromatography.htm
www.shsu.edu/~chm_mfp/gel_filtration_chromatography.htm
analytical gel filtration
chromatography. A preliminary abstract .... gel filtration chromatography of
S100B(PP) in the absence of CaZ+ (10 pM subunit ...
www.proteinscience.org/cgi/reprint/6/7/1577.pdf
www.proteinscience.org/cgi/reprint/6/7/1577.pdf
Nature Protocols is an interactive
online resource for all laboratory ... and characterization of transcribed RNAs
using gel filtration chromatography ...
www.nature.com/nprot/journal/v2/n12/abs/nprot.2007.480.html
www.nature.com/nprot/journal/v2/n12/abs/nprot.2007.480.html
General
Protocol 1. Prepare Column for gel filtration chromatography, and ...
Gel Filtration Chromatography. ? separates solutes based on molecular size. ? also called molecular sieve chromatography or size. exclusion chromatography ...
www.tulane.edu/~wiser/methods/handouts/class/09_chrom.pdf
Gel Filtration Chromatography. ? separates solutes based on molecular size. ? also called molecular sieve chromatography or size. exclusion chromatography ...
www.tulane.edu/~wiser/methods/handouts/class/09_chrom.pdf
Gel filtration chromatography. Gel filtration chromatography was used to separate soluble proteins fromvesicle-associated proteins based on the time required for ...
https://www.sciencedirect.com/.../gel-filtration-chromatography
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they are scammers and got what they deserved!
I have also recently received an
email from Jacob Baby, what a name, sounds like one of the Indian scammers from
a chat site trying to get personal info from you! Anyway, he has asked me to
remove the reviews because it is killing their sales, they can no longer take
our hard earned cash and scam the crap out of innocent people. I said that I
would remove the bad reviews but only if they install a full working version of
their search engine script with all modules and plugins, fully working. The
reason for this is because I want people to be able to see a working version of
their software and I can add it to the bad reviews to show that they have fixed
the issues. The reply from Jacob Baby was a straight “NO”, no way are they
going to install a full working version for free, instead they would do it for
50% off the normal price! This just shows that they do not have any confidence
in their own scripts and that they are all still full of bugs. I am not giving
those scammers any more of my money and I hope I can bring them right down and
destroy them. End of the day, InOutScripts.com is a terrible Indian
scamming company which will promise you the world, take your money and then go
AWOL!
Gene therapy
Gene therapy involves supplying a
functional gene to cells lacking that function, with the aim of correcting a
genetic disorder or acquired disease. Gene therapy can be broadly divided into
two categories. The first is alteration of germ cells, that is, sperm or eggs,
which results in a permanent genetic change for the whole organism and
subsequent generations. This “germ line gene therapy” is considered by many to
be unethical in human beings. The second type of gene therapy, “somatic cell
gene therapy”, is analogous to an organ transplant. In this case, one or more
specific tissues are targeted by direct treatment or by removal of the tissue,
addition of the therapeutic gene or genes in the laboratory, and return of the
treated cells to the patient. Clinical trials of somatic cell gene therapy
began in the late 1990s, mostly for the treatment of cancers and blood, liver,
and lung disorders.
Despite a great deal of publicity
and promises, the history of human gene therapy has been characterized by
relatively limited success. The effect of introducing a gene into cells
often promotes only partial and/or transient relief from the symptoms of the
disease being treated. Some gene therapy trial patients have suffered adverse
consequences of the treatment itself, including deaths. In some cases, the
adverse effects result from disruption of essential genes within the patient's
genome by insertional inactivation. In others, viral vectors used for gene
therapy have been contaminated with infectious virus. Nevertheless, gene
therapy is still held to be a promising future area of medicine, and is an area
where there is a significant level of research and development activity.
www.inoutscripts.com celebrities script: No.1 infamous scamming business
I purchased the Inout Celebrities
Script one year ago, but found script was not updated properly. It is still
using PHP 5.4 engine. The site is very slow. What is nore that the script is
not responsive designed. If you ask them for the mobile edition, they will
charge you another $800 as a customization. Finally, I gave up the site.
There are totally 14 bugs and 25
warnings with this Inout Script.
The manager Kumar and its boss Jacob
are cheaters. Finally, they will take the cash and leave you nothing working.
Their support email address, suppoet@inoutscripts.com has been world
widely labeled as phishing scam.
They advertise with overstated words
to induce customers to purchase their so called "clone scripts". We
purchased 13 of them, and just found they were full of errors and bugs. Their
main group members are Jacob, Kumar, Nair, and Saranya. All of them locate at a
small room in Karala , India
If you are the victim of
Inoutscripts, don't hesitate to contact me.
They have other phishing sites:
Phishing site 1.
http://www.inoutscripts.com
Phishing site 2.
http://www.enterspine.com/
Phishing site 3.
https://www.parishcloud.com/
Phishing site
4. https://www.parishcloud.com/
Phishing site
5. https://www.storecave.com/
Phishing site 6. https://www.nesote.com/
Believe me. Don’t purchase any
scripts from Inoutscripts.com. They are liars, cheaters, and scamming.
Inoutscripts.com the infamous India
DNA Modification/Epigenetics
Analysis of Mitotic Checkpoint
Function in Xenopus Egg Extracts
Yinghui Mao
Cold Spring Harb
Protoc 2018; doi:10.1101/pdb.prot099853
Analysis of DNA Methylation in
Mammalian Cells
Paul M. Lizardi, Qin
Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.top094821
Methylation-Specific Polymerase
Chain Reaction (PCR) for Gene-Specific DNA Methylation Detection Paul M.
Lizardi, Qin Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.prot094847
Methyl-Cytosine-Based
Immunoprecipitation for DNA Methylation Analysis
Paul M. Lizardi, Qin
Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.prot094854
High-Throughput Deep Sequencing for
Mapping Mammalian DNA Methylation
Paul M. Lizardi, Qin
Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.prot094862
DNA Bisulfite Sequencing for
Single-Nucleotide-Resolution DNA Methylation Detection
Paul M. Lizardi, Qin
Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.prot094839
Illumina Sequencing of
Bisulfite-Converted DNA Libraries
Paul M. Lizardi, Qin
Yan, and Narendra Wajapeyee
Cold Spring Harb
Protoc 2017; doi:10.1101/pdb.prot094870
Micrococcal Nuclease Digestion
of Schizosaccharomyces pombe Chromatin
Hugh P. Cam and Simon Whitehall
Cold Spring Harb
Protoc 2016; doi:10.1101/pdb.prot091538
Oncogenomics Methods and Resources
Simon J. Furney, Gunes
Gundem, and Nuria Lopez-Bigas
Cold Spring Harb
Protoc 2012; doi:10.1101/pdb.top069229
Detection of Cytosine Methylation in
RNA Using Bisulfite Sequencing
Tim Pollex, Katharina
Hanna, and Matthias Schaefer
Cold Spring Harb
Protoc 2010; doi:10.1101/pdb.prot5505
In Vitro Histone Demethylase Assay
Yu-ichi Tsukada and Keiichi I.
Nakayama
Cold Spring Harb
Protoc 2010; doi:10.1101/pdb.prot5512
Potassium Permanganate Probing of
Pol II Open Complexes
Michael F. Carey, Craig L.
Peterson, and Stephen T. Smale
Cold Spring Harb
Protoc 2010; doi:10.1101/pdb.prot5479
Amplification of Bisulfite-Converted
DNA for Genome-Wide DNA Methylation Profiling Jon Reinders
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5342
In Vivo DNase I, MNase, and
Restriction Enzyme Footprinting via Ligation-Mediated Polymerase Chain Reaction
(LM-PCR)
Michael F. Carey, Craig L.
Peterson, and Stephen T. Smale
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5277
In Vivo Dimethyl Sulfate (DMS)
Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR) Michael
F. Carey, Craig L. Peterson, and Stephen T. Smale
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5278
Chromatin Immunoprecipitation (ChIP)
Michael F. Carey, Craig L.
Peterson, and Stephen T. Smale
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5279
Native Chromatin Preparation and
Illumina/Solexa Library Construction
Suresh Cuddapah, Artem
Barski, Kairong Cui, Dustin E. Schones, Zhibin Wang, Gang
Wei, and Keji Zhao
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5237
Reconstitution of Nucleosomal Arrays
Using Recombinant Drosophila ACF and NAP1Craig L. Peterson
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5114
Purification of
Recombinant Drosophila ACF
Craig L. Peterson
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5115
Purification of
Recombinant Drosophila NAP1
Craig L. Peterson
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5116
Combined 3C-ChIP-Cloning (6C) Assay:
A Tool to Unravel Protein-Mediated Genome Architecture
Vijay K. Tiwari and Stephen B.
Baylin
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5168
Chromosome Conformation Capture
Nathan F. Cope and Peter Fraser
Cold Spring Harb
Protoc 2009; doi:10.1101/pdb.prot5137
Chicken Erythrocyte Histone Octamer
Preparation
Craig L. Peterson and Jeffrey
C. Hansen
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot5112
Salt Gradient Dialysis
Reconstitution of Nucleosomes
Craig L. Peterson
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot5113
DNA Methylation Analysis of Human
Imprinted Loci by Bisulfite Genomic Sequencing
Vanessa T. Angeles and Renee A.
Reijo Pera
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot5046
Coimmunoprecipitation (co-IP) of
Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis
Berthe Katrine Fiil, Jin-Long
Qiu, Klaus Petersen, Morten Petersen, and John Mundy
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot5049
Mapping Protein Distributions on
Polytene Chromosomes by Immunostaining
Renato Paro
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot4714
DNA Immunoprecipitation (DIP) for
the Determination of DNA-Binding Specificity
Andrea J. Gossett and Jason D.
Lieb
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot4972
Methylated CpG Island
Marcos R. H. Estécio, Pearlly
S. Yan, Tim H-M. Huang, and Jean-Pierre J. Issa
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot4974
In Vitro Histone Methyltransferase
Assay
Ian M. Fingerman, Hai-Ning
Du, and Scott D. Briggs
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot4939
Development of Mammalian Cell Lines
with lac Operator-Tagged Chromosomes
Yuri G. Strukov and Andrew S.
Belmont
Cold Spring Harb
Protoc 2008; doi:10.1101/pdb.prot4903
Micrococcal Nuclease-Southern Blot
Assay: I. MNase and Restriction Digestions
Michael Carey and Stephen T.
Smale
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4890
Micrococcal Nuclease-Southern Blot
Assay: II. Capillary Transfer and Hybridization
Michael Carey and Stephen T.
Smale
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4891
Chromatin Immunoprecipitation (ChIP)
on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications
Alexandre Wagschal, Katia
Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4767
PCR-Based Analysis of
Immunoprecipitated Chromatin
Alexandre Wagschal, Katia
Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4768
Yeast Chromatin Immunoprecipitation
(ChIP) Assay
William P. Tansey
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4642
Denaturing Protein
Immunoprecipitation from Yeast
William P. Tansey
Cold Spring Harb
Protoc 2007; doi:10.1101/pdb.prot4643
Chromatin Immunoprecipitation (ChIP)
of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in
Cultured Cells
Achim Breilingand Valerio
Orlando
Cold Spring Harb
Protoc 2006; doi:10.1101/pdb.prot4560
Mapping DNase-I-hypersensitive Sites
Joseph Sambrook and David W.
Russell
Cold Spring Harb
Protoc 2006; doi:10.1101/pdb.prot3949
Chromatin Immunoprecipitation in
Yeast
David C. Amberg, Daniel J.
Burke, and Jeffrey N. Strathern
Cold Spring Harb
Protoc 2006; doi:10.1101/pdb.prot4177
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