Ultracentrifuge protein concentration

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

Enzyme source (see Note 4) Chinese hamster ovary cells (CHO-Kl, American Type Culture Collection) were transiently transfected via standard DEAE-dextran methods with an expression construct encoding the human ECE-lc protein. The medium was changed to serum-free medium the day after transfection. Two days after transfection, cells were harvested and a crude membrane fraction sedimented by ultracentrifugation (100,000g for 1 h). The pellet was suspended in assay buffer, aliquoted, and frozen at -70°C until assay. Protein concentration was determined by standard methods (e.g., Lowry or BCA). The equivalent of 3 pg total protein was used in each assay well. 

Figure 8. Ultracentrifugal patterns of Pseudomonas cellulases Pictures were taken at 45 minutes after rotation had reached a maximum speed of 55,430 r.p.m. at 17°C. Protein concentration was 0.45% (Cellulase A) and 0.7% (Cellulases B and C) in 1/15M phosphate buffer of pH 7.0. The synthetic boundary cell was employed for Cellulases B and C...

At pH 8 mercuripapain exhibits a complex and interesting behavior in the ultracentrifuge. At high protein concentrations, two sedimenting boundaries are observed (Fig. 7) a lighter component with the Sm.b value of the monomer and a heavier one with Sw.a 8.0 S the latter sedimentation constant corresponds very closely to the value expected for a hexamer. On dilution, the quantity of heavy material diminishes and that of the monomer increases. The behavior is consonant with a system in which 6 moles of papain... 

Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

A) Fractured convex half and (B) concave half of Mab (PhytVSQDG (9 1 mol/ mol) vesicle containing BR (C) convex half and (D) concave half of protein-free vesicle. Bar = 50 nm. Samples were prepared at the initial concentration of 2 mM Mab (Phyt)2, 0.22 mM SQDG and 50pg/ml BR and concentrated by ultracentrifugation in 75 mM K2SO4 buffer solution. 

Each pellet ( dirty microsomal fraction ) was resuspended in 2 mL of ice-cold 1.15 % KCl, collected together and ultracentrifuged again at 100 000 x g for 45 min at 0 °C. The supernatant was discarded and the pellet was rinsed thrice with ice-cold dilution buffer. The rinsed pellet was resuspended in ice-cold 1.15 % KCl to yield a final concentration 20 mg mL of microsomal proteins. The microsomal suspension was immediately aliquoted, frozen and stored at —80 °C. 

A summary of the report follows The problem is to separate proteins. Furthermore, SpinPro should pay particular attention to the purity of the separation. The sample is not negatively affected by sucrose, has a sedimentation coefficient of 16 Svedbergs, and is in liquid form of 3 mL and a concentration of 1% w/w. The protein of interest should be placed 45% from the top of the gradient at the end of the run. Of the gradient concentrations 10-40% and 5-20%, the 10-40% is preferred by the investigator. There are no solvents in the sample that are harmful to the tubes. Finally, from the lab, SpinPro should use the L2-75B ultracentrifuge and the SW 41 Ti rotor, which does not require a speed derating due to its age. 

A method for determining the alteration of the refractive index of a medium as a result of the temperature rise in the path of a beam of coherent light absorbed by the medium. Thermal lensing can also occur with pigmented proteins, and this phenomenon can influence the accuracy of concentration gradient measurements in small aperture flow cuvettes as well as in ultracentrifugation. 

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