You can change your ad preferences anytime. Next SlideShares. You are reading a preview. Create your free account to continue reading.
Sign Up. Upcoming SlideShare. Ultrcentifugation: Basic Training. Embed Size px. Start on. Show related SlideShares at end. WordPress Shortcode. Share Email. Top clipped slide. Download Now Download Download to read offline. Ultracentrifugation Nov. Tapeshwar Yadav Follow. Lecturer of Medical Biochemistry.
Basics of Proteins Chemistry. Medical Terminology of Endocrine System. Basics of Carbohydrates Chemistry. Introduction to biochemistry. Medical Parasitology Laboratory. Medical Microbiology Laboratory. Clinical Hematology Laboratory. Clinical Biochemistry Laboratory. Related Books Free with a 30 day trial from Scribd. Jen Gunter. Related Audiobooks Free with a 30 day trial from Scribd.
Gundry, MD. Permission to Dream Chris Gardner. Single On Purpose: Redefine Everything. Find Yourself First. John Kim. Ultracentrifugation 1. Medical Biochemistry 2. Centrifuge A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. Ultracentrifugation Machine 5. Example; Red cells separated from plasma of blood, nuclei from mitochondria in cell homogenates, one protein from another in complex mixtures.
TYPES 1. Analytical ultracentrifugation:- The aim of Analytical ultracentrifugation is use to study molecular interactions between macromolecules or to analyse the properties of sedimenting particles such as their apparent molecular weight. Preparative ultracentrifugation:- The aim of Preparative ultracentrifugation to isolate and purify specific particles such as subcellular organelles.
There are two types of ultracentrifugation: 7. Analytical ultracentrifugation Two kinds of experiments are commonly performed on these instruments: 1. Sedimentation velocity experiments:- Aim of SVEs to interpret the entire time-course of sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as their size distribution.
Sedimentation equilibrium experiments:- SEEs are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time-independent concentration profile.
Preparative ultracentrifugation It is to isolate specific particles which can be reused 1. The instrument operating parameters include: the temperature, the rotor speed, time after speed is reached before the first scan is taken, the time interval between scans, and how many scans are to be acquired.
For sedimentation velocity analysis, there should be no delay before data are acquired. Likewise, there is no reason to wait between scans, so there should be no interval between scans.
These two parameters scan delay and scan interval should be set to zero in the method for either the Beckman Coulter ProteomeLab or the Aviv-AOS software to maximize the number of data sets available for analysis.
For a sedimentation velocity experiment, one wants to make sure the samples have stabilized at the desired temperature prior to rotor acceleration. For this reason, many people allow the system to stabilize at temperature for an hour or so before acceleration. For high accuracy work, it is desirable to calibrate the XLI temperature sensor Liu and Stafford, Choosing the correct rotor speed for a sedimentation velocity experiment depends what you want to know about your sample, what the expected component size distribution is, and which optical systems will be used.
These considerations lead to competing needs. However, with modern global analysis software it is also beneficial to obtain a large number of scans. Thus, lower rotor speeds are required if components of interest are very large with large sedimentation coefficients. Also, the absorbance optics have long scan times and when scanning multiple samples and wavelengths it may be useful to reduce rotor speeds.
Although there is no simple formula for optimizing the rotor speed, we can use the definition of the sedimentation coefficient, equation 2 to determine reasonable rotor speeds. It should take a boundary at least 2 hours to sediment the full length of the cell 1.
Based on this criterion, the maximum recommended rotor speeds for various sedimentation coefficients are presented in Table 3. In addition, when using the absorbance system it is necessary to consider the longer scan times and adjust the rotor speed so that at least 30—40 scans are recorded during the movement of the boundary across the cell.
Methods for analysis of SV experiments have evolved rapidly in recent years and many alternative approaches and software packages are available. Here, we will outline an approach that we have found useful in the initial stages of analyzing an unknown system. At the early stages, it is useful to examine the data using methods that require the fewest assumptions about the nature of the system being investigated.
Later, this information can be used to construct models and obtain starting parameters for more detailed analyses. This subtraction removes the systematic noise in the data, which is particular useful for interference data.
It is important to inspect the distributions at multiple loading concentrations to check for reversible interactions. This behavior is diagnostic for slowly reversible interactions and it is important to fully equilibrate such samples prior to AUC analysis.
For homogeneous species or mixtures of noninteracting species, the width of each peak is related to D, and one can fit the distribution to obtain D and thus the molecular mass of each component.
This fitting process can also be useful to determine whether the peak is truly homogeneous. Recent advances have improved the fitting function Philo, and extended the scan range Philo, than can be used in this analysis.
The main advantage of the dcdt method is simplicity. No models are assumed in the analysis. Also, the subtraction and averaging results in noise reduction, allowing lower sample concentrations. The chief disadvantages are the limitations in the number of scans to avoid distortion of the peak shape, and the diffusional broadening of the peaks that can hide heterogeneity. Here, we describe the most basic implementation of the c s method. First, the program creates a grid of sedimentation coefficients covering the expected range of interest.
The program then simulates the sedimentation boundaries for each point using a numerical solution of the Lamm equation. Finally, the data are fit to a sum of these Lamm solutions using a least-squares fitting procedure to define the concentration of each species in the grid.
During this process, the systematic noise of the baseline time invariant noise and the vertical displacements jitter and integral fringe jumps are removed by treating them as additional linear fitting parameters. For the c s models, one can also check whether the model of a sum of noninteracting species provides a good fit to the experimental data.
A poor fit can indicate reversible interactions. Although the c s distribution can be converted to a distribution of molar masses c M distribution , the derived masses will only be accurate if there is one dominant species present or if all the species have equal frictional ratios.
The main advantages of the c s method are the excellent resolution and sensitivity. The c s method is thus very useful for characterizing homogeneity and quantitating impurities and aggregates. The main disadvantage of this approach is that it assumes a noninteracting mixture and particular care must be exercised in the analysis of self- or hetero-associating systems where the resulting distributions are developed from an incorrect model.
Nonetheless, for a system undergoing rapid association and dissociation, the distributions are reminiscent of those expected by limiting models 57 , and useful semi-quantitative information may be extracted 53, For interacting systems undergoing reactions on the timescale of the SV experiment, peaks in the c s distribution may not correspond to true molecular species Dam et al.
Provided that the c s distribution is a good fit to the data, it is always feasible to extract thermodynamic parameters from the data by integration of the distribution and analyzing the dependence of weight average sedimentation coefficients on the loading concentrations Correia, ; Correia et al. The only requirement for this analysis to be accurate is that all association reactions are at equilibrium prior to the start of sedimentation.
This criterion may be met by incubating the sample dilutions a sufficient amount of time e. The van Holde — Weischet approach van Holde and Weischet, is also used for the initial, qualitative analysis of SV experiments. Because sedimentation is proportional to the first power of time whereas diffusion is proportional to the square root of time, graphic extrapolation of the boundary to infinite time yields an integral sedimentation coefficient distribution, G s in which the diffusional contribution has been removed.
Recent advances have extended this method for the analysis highly heterogeneous systems. Demeler and van Holde, Although the information obtained from the model-free approaches may be enough to answer the relevant questions about the macromolecular system being studied, we often find it useful to analyze the system using model-dependent procedures.
For analysis of mixtures, the goal is usually to obtain the concentration, sedimentation coefficient and mass of each species. These parameters are recovered with greater precision by using a fitting model of a mixture of several discrete species rather than continuous distribution approaches.
The absence of systematic deviations in global fit also confirms that there are no mass action reactions over the concentration range examined. For systems that contain a mixture of well-defined discrete species and poorly resolved aggregates or low molecular weight impurities, it can be useful to fit the data to a hybrid c s -discrete species model in SEDPHAT where the poorly resolved material is accounted for in the continuous distribution.
However, SV may be the only feasible approach for systems that are intrinsically unstable or that do not come to equilibrium in SE experiments. For interacting systems, the boundaries do not generally correspond to discrete species. Because the concentrations are changing throughout the cell during sedimentation, particularly where there are boundaries, the species composition is continuously varying due to the mass action equilibria.
Consequently, the apparent sedimentation coefficients and boundary shapes are complex functions of the sedimentation coefficients of the species participating in the equilibrium, their concentrations, and the equilibrium and kinetic constants governing their interactions Cann, ; Dam and Schuck, ; Gilbert and Jenkins, As alluded to above, the traditional approach to analyzing interacting systems by SV is to measure weight average sedimentation coefficients as a function of loading concentrations Correia, ; Correia et al.
An advantage of this method is that the weight average sedimentation coefficient is a thermodynamically valid parameter that is determined the sample composition in the plateau and is independent of the kinetics of the interactions, provided that the sample is at equilibrium prior to sedimentation.
Examples of this approach can be found in studies of CMV protease dimerization Cole, and the complex association reactions of tubulin Correia, ; Sontag et al.
When compared, this approach gives comparable results to those obtained using weight-average analysis Sontag et al. Some recent examples of direct boundary analysis to define the energetics of associating systems can be found in Connaghan-Jones et al. The big advantage of sedimentation equilibrium, SE, is that it removes all hydrodynamic effects, so that purely thermodynamic analysis is possible.
The requirements for sample purity and homogeneity are much stricter for SE measurements that for velocity experiments. In the latter case, the boundaries associated with each species separate during the sedimentation run so that it is possibly to isolate contaminants from the species of interest. In contrast, different species are incompletely fractionated in an SE gradient. Furthermore, as shown below, fitting SE concentration gradients requires deconvolution of multiple exponential functions, which is a challenging mathematical operation that becomes increasingly difficult with larger numbers of species.
There are three commercially available centerpiece styles that are commonly used when conducting SE experiments. The choice of which style to use will be determined by the information that is being sought.
The short-column centerpiece has 8 channels which can hold four sample-reference pairs. The standard long-column centerpiece has 6 channels, which can hold three sample-reference pairs Figure 1B. Long-column experiments are useful for accurately determining molecular weights, self-associations, hetero-associations, etc. A version of the 6 channel centerpieces is available that, along with a custom cell housing, allows the cells to be loaded and unloaded without disassembly Ansevin et al.
First assemble the external loading cell according to specifications typically sealed at between and inch-pounds of torque. Centrifuge the cells at the maximum speed that will be used during your experiment for at least 1 hour.
Stop the run, remove the cells, and re-torque them to specifications. Place the cells back in the rotor and centrifuge them at the same speed as before for another hour.
In our experience three or four cycles is sufficient to bring the cell into a stable state. To acquire the blank, the cells are filled with water and run at the same temperature and rotor speeds as will be used during the experiment. At each rotor speed, scans are acquired every 5 minutes or so until no changes in the fringe patterns are apparent. After the blanks have been acquired the water is removed, the cells dried, and the samples loaded.
Because they do not need disassembly, the blank correction from external loader cells above can result in fold lower noise Ansevin et al. Specialized methods for washing the external loading cells without disassembly have been described Ansevin et al. An automated cell washer recently became available Spin Analytical, NH. Also, Beckman Coulter produces centerpieces that facilitate cell cleaning by incorporating two holes per sector.
In order to characterize a system over a wide concentration range, different sample loading concentrations must be used. It is recommended that , and dilutions be used with the 6-channel cells, and , , , dilutions be used in the 8-channel cells. This way, advantage will be taken of the gravitational field to concentrate the more dilute samples while minimizing the concentration gradients in the highest concentration sample.
A layer of dense, colorless fluid should be used to create an artificial base of each sample. This layer allows data acquisition at the highest concentration region with less interference from reflections from the centerpiece base. The recommended fluid is FC 3M, Inc. It has been found that certain proteins e.
Thus, while it is generally inert, it is worthwhile checking to make sure FC is compatible with the solution components. Other than the cells that are employed, there is no change in the instrumentation from SV for SE experiments. However the operating parameters are different.
Unlike SV, it is usually not critical to allow temperature equilibration prior to starting the rotor spinning. It is important to collect data at multiple loading concentrations and rotor speeds to assess if the sample is homogeneous, if mass action-driven self-association is occurring, or if thermodynamic nonideality is significant.
The complete data set can be used subsequently in global curve fitting programs to obtain the most precise parameters from the data. Many researchers perform sedimentation equilibrium experiments using rotor speeds that are too low e. A typical experimental protocol will produce data at three or four rotor speeds using 1.
In combination with the recommended cell loading described above, this protocol will produce data over a very broad concentration range that will enhance the reliability of the analysis. The experimental protocol must go from lowest to highest rotor speed. If a lower rotor speed is used after a higher one, the system will not reach equilibrium in a reasonable time Roark, The time to achieve equilibrium is dependent on a number of experimental factors, including the mass and shape of the particle, the solvent viscosity and the distance between the meniscus and the base column height.
In particular, the equilibrium time is proportional to the square of column height. Although theoretical expressions are available for the simplest systems van Holde and Baldwin, , the actual time to equilibrium may be extended by slow association and dissociation rates and other factors.
Thus, the approach to equilibrium is often monitored experimentally by taking the difference between successive scans and looking for the absence of systematic deviations. These programs do least-squares comparison of the scans allowing for displacements in the vertical and horizontal directions. The RMS deviations decrease as a function of time until at equilibrium they reach a constant level corresponding to the noise level in the data. Although the equilibrium method in the Beckman-Coulter XL-I control software allows one to insert a delay prior to recording data, we recommend collecting data immediately at regular 15 — 30 minute intervals to monitor the approach to equilibrium.
When using the absorbance system, we typically record scans using a coarse point spacing of 0. Once equilibrium is achieved, the sample is then scanned using the maximal point spacing of 0. Slow aggregation can cause a loss of material in successive scans and prevent achievement of equilibrium. Other potential problems in equilibrium experiments can include sample hydrolysis or denaturation.
In some cases, problematic samples can be stabilized by altering the buffer composition, temperature or changing the protein construct. However, it may be necessary to reduce the column height to achieve rapid equilibrium or use more rapid techniques, such as SV. There are several ways to analyze SE data. While this method is no longer widely used, it highlights a problem that must be addressed by all analysis methods, namely that one must have an accurate estimate of the concentration.
It is tempting to substitute the absorbance, fringe displacement or fluorescence intensity signal since each of these is proportional to the concentration. Before they can be used, however, it is necessary to adjust the signal to be zero at zero concentration.
This adjustment is accomplished by subtracting a baseline offset. Alternatively, the offsets may be treated as fitting parameters in nonlinear least squares analysis software. With interference data the offsets must be treated as fitting parameters.
Data analysis can be divided into two general methods- molecular weight moment determination and nonlinear least squares fitting. Both of these methods are useful, depending on what information is sought. Molecular weight moments can be determined directly from the data using the ratio of the different concentration moments Harding et al. No model needs to be specified for these calculations, so they are particularly useful for the analysis of complex systems.
Programs are available specifically for calculating molecular weight moments Table 4. Nonlinear least squares analysis of sedimentation data have been performed for over 40 years Johnson et al. Most nonlinear least squares fitting programs directly fit the experimental data to particular models, such as a single ideal species:.
Modern SE analysis software data can incorporate data obtained at multiple rotor speeds using multiple signals for global analysis Table 4.
More complex models are required for analyzing data for associating systems and for systems exhibiting thermodynamic non-ideality Johnson et al. The newer analysis packages are capable of analyzing heteroassociation reactions involving two or more components.
These models involve a large number of adjustable parameters and it is often necessary to constrain the fitting process by incorporating multiple signals Cole, ; Howlett et al.
It should be stressed that SE does not have the resolving power of SV, and reliable analysis of SE data by nonlinear least squares fitting methods requires pure samples free of aggregated material or contaminants. Depending on the size of the aggregates, it may be possible to pellet them while still analyzing the remaining sample. However, the presence of aggregates or contaminants will lead to inconsistencies in the data analysis Thus, it is critical to characterize samples by SV prior to SE experiments.
In some cases, contaminants or aggregates identified by the SV measurements can be removed by preparative gel filtration prior to SE analysis. Analytical ultracentrifugation is a versatile and rigorous technique for characterizing the molecular mass, shape and interactions of biological molecules in solution. In particular, the size distribution analysis available with SV is more flexible, is applicable to more chemical systems, spans a much wider range of sizes and provides higher resolution than size exclusion chromatography.
The hydrodynamic information available with SV is complemented by thermodynamic analysis by SE. The availability of interference refractive , absorbance and fluorescence detectors makes AUC applicable to a wide variety of questions in cell biology. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules under physiological conditions.
As we have described above, modern AUC users can choose from a broad array of experimental techniques and data analysis methods, and it can be difficult to decide how to best apply AUC methods when confronted with a new sample.
Although the best strategy will depend considerably on the nature of the sample and the kinds of questions that need to be answered, Figure 2 shows a typical workflow that we use for characterizing a new sample by AUC. It is strongly recommended that new samples are first analyzed by SV at several concentrations.
These measurements are crucial for deciding whether the sample is homogeneous and suitable for more detailed analysis. If contaminants or aggregates are present that differ appreciably in size from the molecule of interest, the sample can often be purified by preparative gel filtration. In fact, we typically gel filter sample prior to AUC analysis. It should also be noted that although dynamic light scattering DLS lacks the resolving power of AUC, it a fast and sensitive method to determine whether aggregates are present and we often use DLS as a quality-control step prior to AUC.
Typical workflow for a analytical ultracentrifugation analysis of an unknown sample. For details see the text. The next step is to determine whether the sample undergoes reversible, mass-action association. For a non-interacting system, the SV data can be analyzed to obtained s and D for the species of interesting and the data can be interpreted to obtain the molar mass and shape parameters.
It should be stressed that this analysis cannot be done for an interacting system: here, more sophisticated analysis is required to measure the sedimentation coefficients of the interacting species and to define the kinetics and thermodynamics of the interaction.
If the system is stable, the interaction can be characterized by SE. Similarly, for a stable noninteracting system, reliable measurement of the molar mass and stoichiometry can be obtained by SE. Throughout this review we have described a large number of data analysis packages available for both SV and SE.
Table 4 lists the websites where this software may be obtained along with references describing the analysis algorithms and their applications. Table 4 also includes a number of utility programs that perform useful calculations or graphics. National Center for Biotechnology Information , U. Methods Cell Biol. Author manuscript; available in PMC Jul James L.
Jeffrey W. Thomas M. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Methods Cell Biol. See other articles in PMC that cite the published article. Abstract Analytical ultracentrifugation AUC is a versatile and powerful method for the quantitative analysis of macromolecules in solution.
Introduction For over 75 years, analytical ultracentrifugation AUC has proven to be a powerful method for characterizing solutions of macromolecules and an indispensable tool for the quantitative analysis of macromolecular interactions Cole and Hansen, ; Hansen et al. Types of problems that can be addressed Analytical ultracentrifugation provides useful information on the size and shape of macromolecules in solution with very few restrictions on the sample or the nature of the solvent. Basic Theory Mass will redistribute in a gravitational field until the gravitational potential energy exactly balances the chemical potential energy at each radial position.
Sedimentation Velocity We can understand a sedimentation velocity experiment by considering the forces acting on a molecule during a sedimentation velocity experiment. Sedimentation Equilibrium When the centrifugal force is sufficiently small, an equilibrium concentration distribution of macromolecules is obtained throughout the cell where the flux due to sedimentation is exactly balanced by the flux due to diffusion.
Dilute Solution Measurements For dilute solutions containing a single macromolecular component, detailed information is available from both sedimentation equilibrium and sedimentation velocity analysis Cole and Hansen, ; Hansen et al.
Concentrated and Complex Solutions If a solution contains a single macromolecular component at high concentration, then one may use sedimentation equilibrium analysis to extract thermodynamic information.
Instrumentation and Optical Systems The analytical ultracentrifuge is similar to a high-speed preparative centrifuge in that a spinning rotor provides a gravitational field large enough to make molecular-sized particles sediment.
Table 1 Capabilities of optical systems. Absorbance Interference Fluorescence Sensitivity a 0. Open in a separate window. For the current interference optical system, this ratio is closer to Table 2 Strengths and weaknesses of optical systems. Characteristic Absorbance Interference Fluorescence Radial resolution a 20—50 10 20—50 Scan time b 60— 1—10 60—90 When to use c Selectivity Sensitivity Non-dialyzable components Solvent absorbs light Solute does not absorb light Accuracy needed Short solution columns Selectivity Sensitivity Small sample quantities Non-dialyzable components.
The time listed for the fluorescence system is the time needed to scan all of the samples Laue, Since the Rayleigh interference optics relies on differences in the refractive index of the sample and reference solutions, it provides no selectivity. The interference optics require that samples are at dialysis equilibrium with the reference solution; hence, they should not be used for samples containing non-dialyzable components e. Absorbance Absorbance is the most frequently used detector for the analytical ultracentrifuge Laue, Fluorescence The fluorescence optical system is the most recent addition to the XLI.
Sedimentation Velocity A. Instrument Operation and Data Collection Sedimentation velocity experiments are carried out in two channel cells with sector shaped compartments Figure 1 in order to prevent convection, which would occur if the cell walls were not parallel to radial lines. Figure 1. Table 3 Maximum rotor speeds for sedimentation velocity experiments. S a M b approximate RPM c 10 , 55, 15 , 50, 30 1,, 30, 90 5,, 20, 25,, 10, However, acquiring absorbance data at multiple wavelengths will greatly increase scan times, thus decreasing the number of scans acquired at a particular wavelength over the course of an experiment.
For experiments requiring multi-wavelength scanning, one may wish to spin at a lower rotor speed. If the molecules are asymmetric or a highly solvated, then a higher molecular weight will correspond to a given sedimentation coefficient. Be sure the maximum speed rating for the centerpiece is not exceeded. Data Analysis Methods for analysis of SV experiments have evolved rapidly in recent years and many alternative approaches and software packages are available. Sedimentation Equilibrium The big advantage of sedimentation equilibrium, SE, is that it removes all hydrodynamic effects, so that purely thermodynamic analysis is possible.
Instrument Operation and Data Collection There are three commercially available centerpiece styles that are commonly used when conducting SE experiments. Monitoring Approach to Equilibrium The time to achieve equilibrium is dependent on a number of experimental factors, including the mass and shape of the particle, the solvent viscosity and the distance between the meniscus and the base column height.
Data Analysis There are several ways to analyze SE data. Table 4 AUC analysis programs and utilities. Discussion and Summary Analytical ultracentrifugation is a versatile and rigorous technique for characterizing the molecular mass, shape and interactions of biological molecules in solution.
Figure 2. Anal Biochem. Calculation of the partial specific volume of proteins in concentrated salt and amino acid solutions. Methods Enzymol. Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles.
Biophys J. Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data. Hydrodynamic bead modeling of biological macromolecules. Interacting Macromolecules.
Academic Press; New York: Biophysical Chemistry. Part II. Freeman and Co; San Francisco: Analysis of heterogeneous interactions. Characterization of human cytomegalovirus protease dimerization by analytical centrifugation. J Biomolecular Techniques. Hydrodynamic analysis of the human progesterone receptor A-isoform reveals that self-association occurs in the micromolar range.
Analysis of weight average sedimentation velocity data. Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles. Sedimentation velocity analysis of heterogeneous protein-protein interactions: sedimentation coefficient distributions c s and asymptotic boundary profiles from Gilbert-Jenkins theory. Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c s Biophys J.
Modern Analytical Ultracentrifugation: Techniques and Methods. Royal Society of Chemistry; Cambridge: Determination of molecular parameters by fitting sedimentation data to finite-element solutions of the Lamm equation. Sedimentation velocity analysis of highly heterogeneous systems. Automatic Measurement of Interefernce Photographs for the Ultracentrifuge. Progr Colloid Polym Sci. Measurement of partial specific volume by sedimentation equilibrium in H2O-D2O solutions.
Analytical ultracentrifugation in a Gibbsian perspective. Biophys Chem. Foundations of ultracentrifugal analysis. Wiley; New York: Calculation of hydrodynamic properties of globular proteins from their atomic-level structure.
Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Mutational analysis of the energetics of the GrpE. DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation. J Mol Biol. Boundary problems in the sedimentation and electrophoresis of complex systems in rapid reversible equilibrium. Analytical ultracentrifugation of complex macromolecular systems. Analytical Ultracentrifugation in Biochemistry and Polymer Science.
Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis. J Biol Chem. Defining the structure and stability of macromolecular assemblies in solution: the re-emergence of analytical ultracentrifugation as a practical tool.
Analytical ultracentrifugation for the study of protein association and assembly. Curr Opin Chem Biol. Specific refractive index increments. In: Huglin MB, editor.
Light scattering from polymer solutions. Academic Press: New York; Analysis of data from the analytical ultracentrifuge by nonlinear least squares techniques. Analysis of transport experiments using pseudo-absorbance data. University of New Hampshire. Choosing which optical system of the optima XL-I analytical centrifuge to use. Progress in Colloid and Polymer Science. Springer; Berlin: Sedimentation Equilibrium as Thermodynamic Tool.
Short column sedimentation equilibrium analysis for rapid characterization of macromolecules in solution. Computer-aided interpretation of analytical sedimentation data for proteins. Modern Applications of Analytical Ultracentrifugation. Ann Rev Biophys Biomol Struct. Modern analytical ultracentrifugation in protein science: A tutorial review. Protein Sci. The calculation of partial specific volumes of proteins in guanidine hydrochloride.
Arch Biochem Biophys. Partial specific volumes and interactions with solvent components of proteins in guanidine hydrochloride. Fluorescence detection for the XLI analytical ultracentrifuge. X-ray and neutron scattering analyses of hydration shells: a molecular interpretation based on sequence predictions and modelling fits. An improved function for fitting sedimentation velocity data for low- molecular-weight solutes.
Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques. Improving sedimentation equilibrium analysis of mixed associations using numerical constraints to impose mass or signal conservation.
Measuring sedimentation, diffusion, and molecular weights of small molecules by direct fitting of sedimentation velocity concentration profiles. Modern Analytical Ultracentrifugation. Birkhauser; Boston: A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions.
Re-examining the oligomerization state of macrophage migration inhibitory factor MIF in solution. Calculation of partial specific volumes of proteins in 8 M urea solution.
Determination of protein molecular weight in complexes with detergent without knowledge of binding. J Am Chem Soc. Ultracentrifuge Studies with Rayleigh Interference Optics. General Applications. J Phys Chem. A General Procedure. Tracer sedimentation equilibrium: a powerful tool for the quantitative characterization of macromolecular self- and hetero-associations in solution.
Biochem Soc Trans. Characterization of heterologous protein-protein interactions using analytical ultracentrifugation. Sedimentation equilibrium techniques: multiple speed analyses and an overspeed procedure.
Studies of self-associating systems by equilibrium ultracentrifugation. Ann N Y Acad Sci. Ultracentrifugation in Biochemistry. National Academy of Sciences; Washington, D. C: A model for sedimentation in inhomogeneous media. Dynamic density gradients from sedimenting co-solutes. On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Sedimentation analysis of noninteracting and self-associating solutes using numerical solutions to the Lamm equation.
Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Direct sedimentation analysis of interference optical data in analytical ultracentrifugation.
A brief introduction to the analytical ultracentrifugation of proteins for beginners. Analytical Ultracentrifugation. A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems. Analysis of reversibly interacting macromolecular systems by time derivative sedimentation velocity. Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile.
Analysis of heterologous interacting systems by sedimentation velocity: curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants. HIV Rev self-assembly is linked to a molten-globule to compact structural transition.
Physical Chemistry of Macromolecules.
0コメント