Sucrose gradient analysis

Light sucrose solution 10% (w/w) sucrose (Serva) 20 mM Tris-HCl, pH 8.0 5 mM MgCl2 140 mM KC1 

Swing-out bucket rotor (e.g. Beckman SW41 Ti rotor—k factor  

In the following order Start the magnetic stirrer, open the gradient mixer outlet, open the valve between the chambers, and start the peristaltic pump. Fill the tubes and take care to stop the pump before air is introduced into the bottom of the tube. Withdraw the capillary carefully. 

Place the filled tubes in prechilled SW41 buckets and leave on ice while the sample is prepared.0 

Apply the sample gently to the meniscus of the gradient.d Weigh 

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Fig. 14. Polysomal mRNP from rat liver. A. Sucrose gradient analysis of the polyribosomes preparation treated with EDTA. Labeling period, 40 minutes. Centrifugation was at 21.000 rpm for 16 hours. —A A Cts/min -------, absorbance. B. CsCI equilibrium gradient analysis of the poly-...

In Fig. 8 a sucrose gradient analysis is shown, demonstrating very nicely that the extent of stimulation by eRF on the 40 S initiation complex formation (left panel) is completely abolished by the action of HRI as is depicted in the right panel. Furthermore one can notice that without eRF a low level of initiation is maintained (see also Figs. 5 and 7), which fits neatly the observations made in the crude lysate system. In the presence of hemin a high initiation rate exists in the absence of hemin, when HRI is activated, the system is forced down to a low initiation rate. In the lower panel of Fig. 8 the amounts of Met-tRNA present on 40 S subunits in this experiment are summarized. The data on 80 S initiation complex formation are in essence similar and need no further discussion. 

It is now well established that DNA strand breaks are activators of poly(ADP-ribose) polymerase. By alkaline sucrose gradient analysis, we have been unable to attribute the activation of polymerase due to hyperthermia to an accumulation of DNA strand breaks [4]. However, it is possible that hyperthermia results in a small number of DNA strand breaks that are undetectable by alkaline sucrose gradient analysis yet produce a powerful activation of the polymerase. Conversely, it is also... 

Figure 3 Sucrose gradient analysis of cytoplasmic extracts of infected and minfected cells. Cells were harvested 3 h or 5 h after mock adsorption or virus adsorption. The details of this experiment are reported in Colby et al (9).

Sucrose gradient analysis of pulse-labeled RNA extracted from infected cells (Hay et al., 1966 Flanagan, 1967) revealed a decline in the rate of synthesis of rapidly labeled high molecular weight RNA (precursors of rRNA and probably mRNA). 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

Fig. 10.21. Comparative analysis of nucleolar preribosomes by electrophoresis and sedimentation. Cells were labeled for 30 min with ( H)uridine (0.3 /jCi/ml, 1.2 /iM uridine, 4 x 10 cells/ml) and nucleoli prepared from which the preribosomes were extracted. A) one part of these preribosomes with some cytoplasmic ribosomes as markers were analysed by electrophoresis on a 2.2% uniform polyacrylamide gel B) Another part was analysed by sedimentation on a 5-20% sucrose gradient in EDTA-buffer...

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