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The Analytical Ultracentrifuge Facility is located in the laboratory of Dr. John "Jack" Correia in the Department of Biochemistry, G208-G209, in the Guyton Research Building. It is directed by Dr. Correia.The facility includes a Beckman XLA Analytical Ultracentrifuge equipped with absorption optics, an AVIV FDS system for fluorescence detection, an Anton Paar DMA 5000 for density measurements, and an Anton Paar AMVm micro viscometer. Information can be requested by calling (601) 984-1522 or e-mailing firstname.lastname@example.org.
All collaborative or service work requires a detailed description of the requested study (see below). Collaborative work assumes authorship on any papers that might arise from these studies. Service for a fee work shall be billed at approximately $500/velocity run (up to three samples plus preliminary analysis); approximately $1,000/equilibrium run (up to three cells plus analysis); ancillary measurements, density or viscosity, $100/determination. These figures may vary depending upon the additonal work and time required to prepare for perform these experiments. More extensive analysis, especially work requiring programming may require additional billing. The cost of running and maintaining a facility has necessitated the need for cost recovery for all collaborative work. Thus each collaborator must agree to pursue funding for the work, either by direct billing for runs or in the form of an NIH subcontract agreement. All publications that result, for collaborative or service work, must reference the UMMC Analytical Ultracentrifuge Center. All arrangements must be made in advance. Once the proposed work is agreed upon a PO # should be provided for billing purposes. Billing shall be handled by UMMC Accounting with payment made to the UMMC Analytical Ultracentrifuge Facility. Note the XLA was purchased with 50% funding from the NSF (BIR9216150) and thus collaborative research may fall under federal guidelines.
Rationale for the study
This should include a brief description of the project and what information you desire from the ultracentrifuge study. In addition any prior ultracentrifuge or biophysical work done on this system should be referenced.
Describe relevant biochemical and physical properties such as solubility, temperature stability, and list values or estimates for physical properties with references when available. For proteins the amino acid composition should be provided so that we can estimate the monomer molecular weight and calculate the partial specific volume, vbar , of the sample. For single or double stranded nucleic acids, information concerning the sequence or basepair composition would be helpful.
Typically samples are provided ready for us to thaw, dilute and run. This, of course, is a goal and not necessarily achievable with every sample. If we must do any preparative manipulations, details must be discuss and agreed upon in advance.
This should include references if appropriate. Sample purity assessed by SDS PAGE for proteins and denaturing agarose gels for DNA are required. The degree of purity is crucial; under the best of conditions 1% or less contaminant is "visible" to us. This depends upon the signal, in this instrument the absorbance, and thus the E*C, or the extinction coefficient at the appropriate wavelength times the concentration. If a 1% contaminant has an E that is 10 times larger than the 99% pure band, then the XLA will "see" it as 10% of the material, etc. Thus, each sample should be accompanied by a gel to access purity.
This should include sample concentration, complete description of solvent and method of equilibration, quantities available and desired recover of sample if possible.
Equilibration into the appropriate buffer is critical for proper thermodynamic characterization of protein-protein, protein-nucleic acid, or ligand-macromolecular interactions. Dialysis or rapid column chromatography into the buffer of choice is required. In addition, the buffers must be relatively nonabsorbing at the wavelengths required for the sample. Thus, if work is to be done at 280 nm, then high concentrations of ATP or GTP are precluded, and must be limited to < 100 uM. High concentration of reducing agents such as DTT or 2ME (> 1 mM) can also be problematic, especially if they are allowed to oxidize at room temperature.
At lower wavelengths organic buffers of EGTA, EDTA mixtures can also be problematic. Thus, buffer absorbance must be checked prior to attempting a run. Absorbance values of buffer alone at the desired wavelength should in general not exceed 0.5 because of the effect it has on the signal/noise. Thus, you should perform spectroscopy, wavelength scans, on all anticipated buffer compositions. The ideal buffer is PO4 and NaCl for ionic strength studies but this is not always possible or desirable. Extinction coefficients for samples are also required if determination of equilibrium constants is desired. For nucleic acids or proteins, these can often be estimated from the composition. (See Analytical Ultracentrifugation Reading List).
As described above, the signal used is absorbance. The light path in a typical cell is 1.2 cm (3 mm cell are also available), and thus using beer's law and the extinction coefficient, the signal per unit macromolecule can be estimated. The ideal signal is between 0.1 and 1.0 OD depending upon the experiment. Signals between 1-2 OD can be problematic due to nonlinearity, but that is testable. This is obviously wavelength dependent. For DNA at 260 nm samples in the 100 nM to 100 uM concentration range are usable. For proteins the aromatic content or cofactor content is important. At 278 nm tubulin has an extinction coefficient of 1.2 and thus we can work with 1 uM or 0.1 mg/ml solutions. At 230-236 nm we can get down to 0.1 uM or 0.01 mg/ml. Note, as described above this is exceedingly buffer dependent. We must work at 50-100 uM GDP/GTP. Above that the signal is diminished too much by buffer absorbance and the signal/noise is unusable. Other buffer components can also be problematic. For sedimentation velocity experiments, we need 500 ul to preferably 600 ul of final sample (after appropriate dilution) per cell (approximately 1/4 of that for 3 mm centerpieces but at higher OD to satisfy the signal requirements); up to three cells can be run simultaneously. Depending upon the question, experiments as a function of concentration are essential to understanding the system. Comparable amounts of buffer are required to blank. For conventionally equilibrium experiments 130 ul of final sample and buffer are required per channel; up to three channels per cell and three cells can be run simultaneously. Thus nine sample-matching buffer pairs can be run simultaneously. Note it is very important for the samples to be properly equilibrated with the buffer. Other methods require smaller amounts, ie. short column, but also higher concentrations. Samples should be accompanied by 25 - 50 ml of the equilibration buffer. This is for diluting samples but also for measuring the density and viscosity for coverting data to S20,w values to Molecular Weights. Details can and should be discussed in advance.
Any materials that are radioactive, contain viral contaminants or other biohazardous materials must be identified. We reserve the right to refuse to work with biohazardous material. In addition, some buffer materials can be hazardous to the cell components and must be avoided. Certain organic solvents will dissolve or soften the centerpieces. Some salts like F- or fluoride anion at low pH will etch and damage quartz windows.
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