Nuclear extracts chromatography

Horwitz EP, Dietz ML, Fisher DE (1991) Separation and preconcentiation of strontinm from biological, environmental, and nuclear waste samples by extraction chromatography nsing a crown ether. Anal Chem 63 522-525... 

Nuclear extracts can be fractionated by chromatography on DEAE-cellulose to give three peaks of RNA polymerase activity (the use of column chromatography is explained in chapter 6). These three peaks correspond to three different RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent cation concentrations, and sensitivity to the mushroom-derived toxin a-amanitin. The three polymerases and some of their properties are summarized in table 28.4. 

The SETFICS process (Solvent Extraction for Trivalent /-elements Intragroup Separation in CMPO-Complexant System) was initially proposed by research teams of the former Japan Nuclear Cycle Development Institute (JNC, today JAEA) to separate An(III) from PUREX raffinates. It uses a TRUEX solvent (composed of CMPO and TBP, respectively dissolved at 0.2 and 1.2 M in -dodecane) to coextract trivalent actinides and lanthanides, and a sodium nitrate concentrated solution (4 M NaN03) containing DTPA (0.05 M) to selectively strip the TPEs at pH 2 and keep the Ln(III) extracted by the TRUEX solvent (239). However, the DFs for heavy Ln(III) are rather poor. An optimized version of the SETFICS process has recently been proposed as an alternative process to extraction chromatography for the recovery of Am(III) and Cm(III) in the New Extraction System for TRU Recovery (NEXT) process. NEXT basically consists of a front-end crystallization of uranium, a simplified PUREX process using TBP for the recovery of U, Np, and Pu, and a back-end Am(III) + Cm(III) recovery step (240, 241). 

Arisaka, M., Watanabe, M., Kimura, T. 2007. Separation of actinides(EH) from lanthanides(III) by extraction chromatography using new N,N -dialkyl-N,N -diphenylpyridine-2,6-dicarboxyamides. Global 2007 Advanced Nuclear Fuel Cycles and Systems, September, Boise, ID. 

Horwitz, E. P., Dietz, M. L., Chiarizia, R., Diamond, H., Maxwell, S. L., and Nelson, M. R., Separation and preconcentration of actinides by extraction chromatography using a supported liquid anion exchanger Application to the characterization of high-level nuclear waste solutions, Anal. Chim. Acta, 310, 63-78, 1995. 

Actinides found as environmental contamination in mosses collected from a bog in the eastern Italian Alps were analyzed after their chemical separation by extraction chromatography (deposited on steel targets) with respect to isotope ratios and their concentration was determined by LA-ICP-MS. Moss samples were contaminated with a variety of actinide isotopes. The detection limits for actinides were determined as 3.6-7.2 x 10 gg for " Am and respectively. The Pu/ Pu isotope ratio (0.212 0.003) was almost constant within experimental error for all samples investigated. Pu contamination in moss samples was mainly the result of global fallout after nuclear weapons tests. " Am was found at the 2x 10 " gg level. This example demonstrates that mosses can be used as bioindicators for environmental contamination. ... 

Researchers identified hnRNP proteins by first exposing cultured cells to high-dose UV irradiation, which causes covalent cross-links to form between RNA bases and closely associated proteins. Chromatography of nuclear extracts from treated cells on an oligo-dT cellulose column, which binds RNAs with a poly(A) tail, was used to recover the proteins that had become cross-linked to nuclear polyadenylated RNA. Subsequent treatment of cell extracts from unlrradiated cells with monoclonal antibodies specific for the major proteins identified by this cross-linking technique revealed a complex set of abundant hnRNP proteins ranging in size from 34 to 120 kDa. 

Rosenfeld and Kelly (1) purified Nuclear Factor-I (NF-I), a Hela cell protein that enhances initiation of adenovirus DNA replication vitro, by affinity chromatography on a specific DNA-cellulose column. The column was prepared by adsorption to cellulose of a plasmid engineered to contain multiple (88) copies of the binding site for NF-1. NF-1 was purified from a Hela cell nuclear extract by chromatography on Bio-rex 70, coli DNA-cellulose, and two sequential passes on the specific DNA affinity column. 

For many small-scale studies, it is unnecessary to purify a DNA-binding factor to complete homogeneity. Instead, over a hundred-fold enrichment of the factor can be achieved by chromatography of one or several ml of a crude nuclear extract directly on a <1 ml affinity column. Conversely, the flow-through from the column is a specific depletion of the DNA-binding factor from the extract. An example of such an enrichment of heat shock activator protein is shown in Figure 3. 

Fifteen trace elements were determined at ng/g mass fraction levels in high purity Sn by Kolotov et al. (1996) using extraction chromatography on Teflon-supported tributylphosphate (TBP). Tin, together with Sb and In radionuclides, which are formed in secondary nuclear reactions of Sn during irradiation, were retained from 6 M HCl on the column. The Na was removed from the eluted trace element fraction by sorption on hydrated antimony pentoxide (HAP). Gas phase impurities of N, C, and O were also analyzed in the same material by radiochemical photon activation analysis via N, C, and detection. 

Our laboratory has been involved in the identification and characterization of the transcription factors for RNA polymerase I (Pol I)-directed transcription of ribosomal RNA genes (rDNA). To this end, we initially isolated and partially purified a transcriptionally active protein complex which contains RNA polymerase I and the essential Pol I transcription factors (1). Such a fraction was obtained from whole ceU extracts (1) or from nuclear extracts (2). Subsequently, we demonstrated that a fraction obtained during chromatography of the cell extract on a heparin sepharose coliunn could prevent nonrandom transcription of cloned rat rDNA in an in vitro system (3). The major protein in this fraction exhibited characteristics of purified poly(ADP-ribose) polymerase. The present report summarizes the properties of this protein, and describes experiments showing the dramatic appearance of accurately initiated transcript in an unfiractionated whole ceU extract or nuclear extract from a tissue foUowing addition of the protein factor. 

In principle, two fundamentally different methods can be applied to solve this task. The first one is determination of the residual concentrations of the fissile nuclides after irradiation and calculation of the burnup from the difference between final and initial values. For this purpose, the uranium and plutonium fraction has to be separated from the fission and activation products and from each other (e. g. by extraction chromatography) subsequently, the concentrations of the individual isotopes, in particular of the fissile isotopes, are analyzed by mass spectrometry. Well-established analytical techniques for performing such analyses are available, so that only small error margins are to be expected in the determination of the concentrations of the isotopes under consideration. However, there are two problems that can potentially cause systematic errors. The first one is the well-known question of the accuracy of results which have been obtained as a difference between two numbers, which limits the accuracy at lower burnup values in particular. The second problem is that the fissile nuclides are not only consumed by nuclear fission but by neutron capture as well in order to avoid systematic errors here, the capture-to-fission ratio valid for the particular irradiation conditions has to be taken into account in the calculation of depletion during irradiation. If one recalls the complicated buildup and decay mechanisms of actinide nuclides during reactor irradiation (see Fig. 3.5.), it is obvious that such correction requires complex calculations. On the other hand, the direct determination of the residual concentration of fissile nuclides is not influenced by errors due to inaccuracies in the fission yields of fission products to be measured nor by migration-induced inho-mogenities in the fuel. 

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-... 

Uniformly labeled 2,4-dichlorophenol- C (purchased from New England Nuclear Corp, Boston, Mass.) was used in the tracer preparation. This provided a label at all carbon positions in the dibenzo-dioxin structure. 2,7-Dichlorodibenzo-p-dioxin- C after initial cleanup by fractional sublimation, contained approximately 5% of an impurity, detected by thin layer chromatography (TLC) which gave mass peaks at 288, 290, 292, and 294 in the mass spectrometer, consistent with a trichloro-hydroxydiphenyl oxide. This is probably the initial condensation product of the Ullman reaction and is most likely 2-(2,4-dichlorophenoxy)-4-chlorophenol. It was removed easily by extractions with aqueous... 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

---