Gradient isokinetic

Pretlow et al. (1978) showed that a shallow gradient of Ficoll (2.7-5.5%) centrifuged at low speed (about lOOOg) for about 100 min, allowed clear, isokinetic separation of spheres differing 2-fold in volume. The density change in the gradient was small (1.017-1.027 g/ml) and viscosity effects negligible. 

Nuclear receptors solubilized from nuclei of pure cultures of rat oligodendroglial and astroglial cells were sedimented in isokinetic sucrose gradients. As illustrated in Fig. 5 the L-[ I]T3 appeared mainly... 

Hgure 5 Gradient shapes (A) linear, (B) exponential, and (C) isokinetic. 

Fig. 4. The effect of eRF on 40 S initiation complex formation. The assay was performed in 25 /xl incubation mixture containing 20 mM HEPES-KOH, pH 7.6, 120 mM KAc, 2 mAf MgA(%, 0.1 mM spermine, 1 mM ATP, 0.4 mM GTP, 5 mM creatine phosphate, 0.05 unit of creatine kinase, 0.05 Ajeo unit of AUG, 1 mM dithiothreitol, 5 pmole PHlMet-tRNA (4500 cpm/pmole), 10 pmole 40 S, 5 pmole eIF-3, 10 pmole eIF-4C, and either 3 pmole eIF-2 or 2.4 pmole eIF-2 eRF and eRF as indicated. After incubating at 37°C for 15 minutes, the incubation mixtures were chilled, and the amount of 40 S complexes formed was determined on 15-32% isokinetic sucrose gradients in 20 mM Tris-HCl, pH 7.6,120 mM KAc, 3 mM MgAcj, and 7 mM 2-mercaptoethanol, that were run at 4 C for 3 hours at 50,000 rpm in a SW 50.1 rotor, fractionated into 18 fractions of 250 and...

Fig. 6. Interaction between poly(ADP-ribose) and chicken erythrocyte core particles. [ P]-poly-(ADP-ribose) was synthesized by purified poly(ADP-ribose) polymerase incubated with calf thymus polynucleosomes and the polymer was extracted as described by Miwa et al. [26]. Core carticles and the polymer were allowed to interact and then centrifuged on a 5-20% (w/v) isokinetic sucrose gradient containing 40 vM NaCl, 10 xM Iris, 0.2 mM EDTA at pH 7.4. Centrifugation was for 14 h at 40,000 rpm (4°C) in a SW41Ti Beckman rotor...

Fig. 7. Effect of ethidium bromide on the sedimentation of nucleoids from cells incubated benzamide. HeLa cells in suspension culture were incubated with 5 mM benzamide. 4 h after ben-zamide addition cells were sedimented, suspended in saline, and applied to isokinetic sucrose gradients (containing bis-benzimide and increasing amounts of ethidium bromide) overlaid with a lysis buffer containing Triton X-100. 15 min later the gradients were centrifuged for 30 min at 16,000 rpm. The sedimentation distance was determined by yisual inspection under UV light. Inset Effect of benzamide on the sedimentation of nucleoids from benzamide-treated cells. HeLa cells were incubated for 4 h with increasing amounts of benzamide and th the nucleoid sedimentation rate was determined in bis-benzimide containing sucrose gradients as described above...

A student prepares an isokinetic gradient for sedimenting DNA. Later in the day he switches his mind and decides to sediment a protein solution. He uses the same gradient. We believe he is in a spot of trouble. Can you explain why ... 

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