Nuclear extract centrifugation

Centrifuge in Beckman JA20 or equivalent for 30 min at 14,000 rpm. The cloudy supernatant is the nuclear extract. 

Clear the nuclear extract after thawing by centrifugation at 10,000 rpm for 1 min. Transfer the supernatant to a new tube keep on ice.b... 

Centrifuge at maximum speed (15,000 x g) for 5 min at 4°C. Collect the supernatant—this is your nuclear extract. Aliquot 5 pi of each sample and measure the protein concentration using the BCA Protein Assay Kit. Store at -80 °C until use. 

It should be noted that sterility is very important when performing the nuclear extraction and first few steps of the TF protocol. Make sure that the water that is being used is RNase-free, DNase-free, and sterile. All centrifuge tubes must be sterile or previously autoclaved. All pipeting and transferring of materials should take place under a sterile hood. The derived nuclear extract is very sensitive to post extraction modifications and should therefore be handled very carefully. Once a sample is extracted and the protein concentration is taken, the sample should be used immediately or else aliquoted and frozen at -80 °C. Also, in order to assure consistency of results between trials, it is beneficial to use aliquots from the same original batch. 

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

Isopycnic centrifugation. If a post-nuclear extract of hypo-tonically shocked rat TDL is fractionated by means of isopycnic centrifugation in an aqueous sucrose density gradient, all of the sedimentable acid hydrolases, including a small part of cathepsin D, band around a modal density of 1.18 (Fig.l). In agreement with the results presented in Table I, most of the cathepsin D activity is recovered in a soluble, unsedimentable form. The other lysosomal acid hydrolases contribute much less unsedimentable activity. 

Figure 1. Distribution of acid hydrolases and protein after isopycnic centrifugation in sucrose gradients of post-nuclear extracts of rat thoracic duct lymphocytes. The shaded block with a density below 1.10 has an arbitrary density interval of 0.1 and represents the position of the sample layer. The dotted line indicates the histogram obtained if enzyme activity or protein were uniformly distributed. Each histogram shows the average of results with standard deviation, and the number of experiments is given between parentheses.

Often the products of nuclear reactions have very short half-lives. This is especially true for the heaviest elements obtained by bombardment of heavy targets with heavy ions. To identify and characterize such short-lived nuclides, fast separations are required solvent extraction techniques are well suited to provide the required fast separations. For example, the SISAK method [68] has been successfully used in conjunction with in-line gas jet separators at heavy ion accelerators to identify short half-life actinide isotopes produced by collision of heavy atoms. The Sisak method involves use of centrifugal contactors, with phase residence times as low as tenths of a second, in conjunction with in-line radiometric detection equipment. 

Fuel. The nuclear fuel cycle starts with mining of the uranium ore, chemical leaching to extract the uranium, and solvent extraction with tributyl phosphate to produce eventually pure uranium oxide. If enriched uranium is required, the uranium is converted to the gaseous uranitim hexafluoride for enrichment by gaseous diffusion or gas centrifuge techniques, after which it is reconverted to uranium oxide. Since the CANDU system uses natural uranium, I will say no more about uranium enrichment although, as I m sure you appreciate, it is a major chemical industry in its own right. 

Chen, J., Tian, G., Jiao, R., Zhu, Y. 2002. Hot test for separating americium from fission product lanthanides by purified Cyanex 301 extraction in centrifugal contactors. Journal of Nuclear Science and Technology S3 325-327. 

Nakahara, M., Sano, Y., Koma, Y., Kamiya, M., Shibata, A., Koizumi, T., Koyama, T. 2007. Separation of actinide elements by solvent extraction using centrifugal contactors in the NEXT process. Journal of Nuclear Science and Technology 44(3) 373-381. 

In this chapter, Section 10.2 gives an overview of the operation of the Argonne centrifugal contactor. Section 10.3 focuses on the design principles for this contactor. Section 10.4 discusses the worldwide applications of this contactor to solvent-extraction processes of interest to the nuclear and other industries. Comparisons with other types of contactors are made throughout the text, and a separate section is devoted to them in Section 10.4. However, because of their widespread use and the author s particular experience with them, the ANL contactor and its variations remain the primary focus. 

The centrifugal contactor was first used to reprocess spent nuclear fuel at the SRS in 1966 (Webster et al., 1969). For almost 40 years, this 18-stage 25-cm SRL contactor was used for the extraction and scrub sections (the A-bank) of the PUREX (plutonium-uranium extraction) process at the SRS. Contactor operation stopped when the facility in which they were housed was shut down in 2003. This 18-stage contactor replaced a 24-stage mixer-settler. Mixer-settlers continued to be used for the rest of the processing, as most of the radiation was removed in the A-bank. The ability to... 

The INET annular centrifugal contactors are being used to partition high-level liquid waste so that the back end of the nuclear fuel cycle can be simplified. In particular, the TRPO process has been developed at INET for this application (Song, 2000), where TRPO is the extractant in the process solvent. Also known as Cyanex 923, TRPO is a trialkyl phosphine oxide that is made commercially by Cytec Industries (formerly American Cyanamid). It has a high affinity for the actinides. Further... 

UREX+ A solvent extraction process for separating the components of used nuclear fuel so that the unreacted fraction can be reused in an Advanced Burner Reactor. Based on the Purex process, which uses tributyl phosphate in n-dodecane, but using multistage, centrifugal contactors. Developed by the Argonne National Laboratory, Chicago, IL, from 2003 and proposed for use by 2014. 

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