preparing-adherent-cells-for-x-ray-fluorescence-imaging-chemical

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Argonne National Laboratory

The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway. Periplasmic extracts show copper-inducible oxidase activity, attributed to the multicopper oxidase function of PcoA. PcoC, a much smaller protein than PcoA, binds one Cu(II) and exhibits a weak electronic transition characteristic of a type II copper center. ENDOR and ESEEM spectroscopy of Cu(II)-PcoC and the (15)N- and Met-CD(3)-labeled samples are consistent with a tetragonal ligand environment of three nitrogens and one aqua ligand "in the plane". A weakly associated S-Met and aqua are likely axial ligands. At least one N is a histidine and is likely trans to the in-plane aqua ligand. The copper chemistry of PcoC and the oxidase function of PcoA are consistent with the emerging picture of the chromosomally encoded copper homeostasis apparatus in the E. coli cell envelope [Outten, F. W., Huffman, D. L., Hale, J. A., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 30670-30677]. We propose a model for the plasmid system in which Cu(I)-PcoC functions in this copper efflux pathway as a periplasmic copper binding protein that docks with the multiple repeats of Met-rich domains in PcoA to effect oxidation of Cu(I) to the less toxic Cu(II) form. The solvent accessibility of the Cu(II) in PcoC may allow for metal transfer to other plasmid and chromosomal factors and thus facilitate removal of Cu(II) from the cell envelope.

Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon.

Zinc, a metal ion that functions in a wide variety of catalytic and structural sites in metalloproteins, is shown here to adopt a novel coordination environment in the Escherichia coli transport protein ZntA. The ZntA protein is a P-type ATPase that pumps zinc out of the cytoplasm and into the periplasm. It is physiologically selective for Zn(II) and functions with metalloregulatory proteins in the cell to keep the zinc quota within strict limits. Yet, the N-terminal cytoplasmic domain contains a region that is highly homologous to the yeast Cu(I) metallochaperone Atx1. To investigate how the structure of this region may influence its function, this fragment, containing residues 46-118, has been cloned out of the gene and overexpressed. We report here the solution structure of this fragment as determined by NMR. Both the apo and Zn(II)-ZntA(46-118) structures have been determined. It contains a previously unknown protein coordination site for zinc that includes two cysteine residues, Cys59 and Cys62, and a carboxylate residue, Asp58. The solvent accessibility of this site is also remarkably high, a feature that increasingly appears to be a characteristic of domains of heavy metal ion transport proteins. The participation of Asp58 in this ZntA metal ion binding site may play an important role in modulating the relative affinities and metal exchange rates for Zn(II)/Pb(II)/Cd(II) as compared with other P-type ATPases, which are selective for Cu(I) or Ag(I).

A new zinc-protein coordination site in intracellular metal trafficking: solution structure of the Apo and Zn(II) forms of ZntA(46-118).

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )<or=-8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli.

The essential transition metal ions are avidly accumulated by cells, yet they have two faces: They are put to use as required cofactors, but they also can catalyze cytotoxic reactions. Several families of proteins are emerging that control the activity of intracellular metal ions and help confine them to vital roles. These include integral transmembrane transporters, metalloregulatory sensors, and diffusible cytoplasmic metallochaperone proteins that protect and guide metal ions to targets. It is becoming clear that many of these proteins use atypical coordination chemistry to accomplish their unique goals. The different coordination numbers, types of coordinating residues, and solvent accessibilities of these sites are providing insight into the inorganic chemistry of the cytoplasm.

Transition metal speciation in the cell: insights from the chemistry of metal ion receptors.

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.

Uptake, distribution, and speciation of selenoamino acids by human cancer cells: X-ray absorption and fluorescence methods.

Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.

Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy.

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies.

&#x3059;&#x3079;&#x3066;&#x306E;&#x751F;&#x6D3B;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x86CB;&#x767D;&#x8CEA;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x3059;&#x3002;&#x5206;&#x96E2;&#x3068;&#x30A2;&#x30DD;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x540C;&#x5B9A;&#x306E;&#x305F;&#x3081;&#x306E;&#x30C4;&#x30FC;&#x30EB;&#x3082;&#x958B;&#x767A;&#x3067;&#x3059;&#x304C;&#x3001;&#x3057;&#x304B;&#x3057;&#x3001;&#x305D;&#x308C;&#x3089;&#x306E;&#x8907;&#x96D1;&#x306A;&#x6DF7;&#x5408;&#x7269;&#x5185;&#x306E;&#x500B;&#x3005; &#x306E;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x540C;&#x5B9A;&#x3068;&#x5B9A;&#x91CF;&#x306E;&#x6709;&#x52B9;&#x5316;&#x306F;&#x3001;windows &#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x306E;&#x7B2C; 2 &#x30B2;&#x30EB;&#x306E;&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x3068;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306E; x &#x7DDA;&#x86CD;&#x5149;&#x30DE;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x3001;&#x6211;&#x3005; &#x304C;&#x958B;&#x767A;&#x3057;&#x3066;&#x3044;&#x308B;&#x5E83;&#x304F;&#x9069;&#x7528;&#x3067;&#x304D;&#x308B;&#x3001;&#x7279;&#x6B8A;&#x306A;&#x88C5;&#x7F6E;&#x306F;&#x3001;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x306E;&#x6E96;&#x5099;&#x306E;&#x305F;&#x3081;&#x3068;&#x3001;&#x5C06;&#x6765;&#x62E1;&#x5F35;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x9AD8;&#x3044;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x30D8;&#x30E2;&#x30B0;&#x30ED;&#x30D3;&#x30F3; &#x30A6;&#x30EC;&#x30A2;&#x30FC;&#x30BC;&#x3001;&#x30BB;&#x30EB;&#x30ED;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30F3;&#x70AD;&#x9178;&#x8131;&#x6C34;&#x9175;&#x7D20;&#x306E;&#x6DF7;&#x5408;&#x7269;&#x306E;&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x306E;&#x5206;&#x96E2;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;

&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x5206;&#x96E2;&#x3068;&#x86CD;&#x5149; x &#x7DDA;&#x30DE;&#x30C3;&#x30D4;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x308B;&#x91D1;&#x5C5E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x691C;&#x51FA;&#x3002;

&#x6240;&#x6709;&#x751F;&#x6D3B;&#x7CFB;&#x7EDF;&#x90FD;&#x53D6;&#x51B3;&#x4E8E;&#x80DE;&#x8D28;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x867D;&#x7136;&#x5DE5;&#x5177;&#x7684;&#x5206;&#x79BB;&#x548C;&#x9274;&#x5B9A;&#x7684;&#x8F7D;&#x8102;&#x86CB;&#x767D;&#x53D1;&#x8FBE;&#x4E86;&#xFF0C;&#x90A3;&#x4E9B;&#x4F7F;&#x9274;&#x5B9A;&#x548C;&#x5B9A;&#x91CF;&#x5206;&#x6790;&#x7684;&#x4E2A;&#x522B;&#x80DE;&#x8D28;&#x5185;&#x590D;&#x6742;&#x7684;&#x6DF7;&#x5408;&#x7269;&#x662F;&#x624D;&#x521A;&#x521A;&#x840C;&#x82BD;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x7684;&#x7535;&#x6CF3;&#x5206;&#x79BB;&#x78B3;&#x9178;&#x9150;&#x9176;&#x3001; &#x94DC;&#x84DD;&#x86CB;&#x767D;&#x3001; &#x8132;&#x9176;&#x548C;&#x4F7F;&#x7528;&#x672C;&#x673A;&#x7684; 2D &#x51DD;&#x80F6;&#x7535;&#x6CF3;&#x548C; x &#x5C04;&#x7EBF;&#x8367;&#x5149;&#x6620;&#x5C04;-&#x9014;&#x5F84;&#x6211;&#x4EEC;&#x6240;&#x53D1;&#x5C55;&#x800C;&#x5E7F;&#x6CDB;&#x9002;&#x7528;&#xFF0C;&#x4E0D;&#x9700;&#x8981;&#x4E13;&#x95E8;&#x7684;&#x8BBE;&#x5907;&#x3001; &#x6837;&#x54C1;&#x5236;&#x5907;&#x548C;&#x53EF;&#x80FD;&#x8981;&#x5728;&#x5C06;&#x6765;&#x53EF;&#x6269;&#x5C55;&#x7684;&#x8840;&#x7EA2;&#x86CB;&#x767D;&#x7684;&#x6DF7;&#x5408;&#x7269;&#x3002;

&#x7535;&#x6CF3;&#x5206;&#x79BB;&#x548C;&#x68C0;&#x6D4B;&#x7684;&#x80DE;&#x8D28;&#x7531; x &#x5C04;&#x7EBF;&#x8367;&#x5149;&#x6620;&#x5C04;&#x3002;

All living systems depend on metalloproteins. Yet, while tools for the separation and identification of apo-proteins are well developed, those enabling identification and quantitation of individual metalloproteins within complex mixtures are still nascent. Here, we describe the electrophoretic separation of a mixture of carbonic anhydrase, ceruloplasmin, urease, and hemoglobin using native 2D gel electrophoresis and X-ray fluorescence mapping-an approach we have developed to be broadly applicable, not require specialized equipment for sample preparation, and likely to be extensible in the future.

Electrophoretic separation and detection of metalloproteins by X-ray fluorescence mapping.

Tous les syst&egrave;mes vivants d&eacute;pendent des m&eacute;talloprot&eacute;ines. Pourtant, alors que les outils pour la s&eacute;paration et l&#39;identification des apo-prot&eacute;ines sont bien d&eacute;velopp&eacute;s, ceux permettant l&#39;identification et la quantification des m&eacute;talloprot&eacute;ines individuels dans des m&eacute;langes complexes sont encore naissants. Nous d&eacute;crivons ici la s&eacute;paration d&#39;un m&eacute;lange de l&#39;anhydrase carbonique, c&eacute;ruloplasmine, ur&eacute;ase et d&#39;h&eacute;moglobine en utilisant l&#39;&eacute;lectrophor&egrave;se 2D native et x-ray fluorescence mapping-une approche d&#39;&ecirc;tre largement applicable, ne n&eacute;cessitent pas d&#39;&eacute;quipement sp&eacute;cialis&eacute; pour la pr&eacute;paration de l&#39;&eacute;chantillon et susceptibles d&#39;&ecirc;tre extensible &agrave; l&#39;avenir, nous avons d&eacute;velopp&eacute; par &eacute;lectrophor&egrave;se.

S&eacute;paration par &eacute;lectrophor&egrave;se et d&eacute;tection des m&eacute;talloprot&eacute;ines de mappage de fluorescence de rayons x.

Tutti i sistemi viventi dipendono metalloproteine. Eppure, mentre gli strumenti per la separazione e l&#39;identificazione di proteine apo sono ben sviluppati, quelli che consente l&#39;identificazione e quantificazione di metalloproteine individuali all&#39;interno di miscele complesse sono ancora nascente. Qui, descriviamo la separazione elettroforetica di una miscela di anidrasi carbonica, ceruloplasmina, ureasi ed emoglobina tramite elettroforesi 2D nativo e raggi x fluorescenza mapping-un approccio abbiamo sviluppato per essere ampiamente applicabili, non richiedono attrezzature specializzate per la preparazione del campione e probabilmente essere esteso in futuro.

Separazione elettroforetica e rilevamento di metalloproteine di mapping di fluorescenza a raggi x.

&#xBAA8;&#xB4E0; &#xC0B4;&#xC544;&#xC788;&#xB294; &#xC2DC;&#xC2A4;&#xD15C; metalloproteins&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xBD84;&#xB9AC;&#xC640; apo&#xB294; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC2DD;&#xBCC4;&#xC744; &#xC704;&#xD55C; &#xB3C4;&#xAD6C;&#xB294; &#xC798; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC2DD;&#xBCC4;&#xACFC; &#xC815;&#xB7C9;&#xC758; &#xBCF5;&#xC7A1; &#xD55C; &#xD63C;&#xD569;&#xBB3C; &#xB0B4;&#xC5D0;&#xC11C; &#xAC1C;&#xBCC4; metalloproteins &#xADF8;&#xB4E4;&#xC740; &#xC544;&#xC9C1; &#xCD08;&#xAE30; &#xC774;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xC804;&#xAE30; &#xC774;&#xB3D9; &#xD0C4;&#xC0B0; &#xD0C8;&#xC218;, ceruloplasmin, urease, &#xAE30;&#xBCF8; 2 &#xCC28;&#xC6D0; &#xC824; &#xC804;&#xAE30; &#xC774;&#xB3D9; &#xBC95; &#xBC0F; x-&#xC120; &#xD615;&#xAD11; &#xB9E4;&#xD551; &#xD55C; &#xC811;&#xADFC; &#xC6B0;&#xB9AC;&#xB294; &#xAD11;&#xBC94;&#xC704; &#xD558; &#xAC8C; &#xC801;&#xC6A9; &#xAC00;&#xB2A5;&#xD55C;, &#xC0D8;&#xD50C; &#xC900;&#xBE44;, &#xADF8;&#xB9AC;&#xACE0; &#xBBF8;&#xB798;&#xC5D0; &#xD655;&#xC7A5; &#xB420; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xD2B9;&#xC218; &#xC7A5;&#xBE44;&#xAC00; &#xD544;&#xC694; &#xD558;&#xC9C0; &#xAC1C;&#xBC1C;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xD5E4;&#xBAA8;&#xAE00;&#xB85C;&#xBE48;&#xC758; &#xD63C;&#xD569;&#xBB3C; &#xBD84;&#xB9AC;&#xC5D0; &#xC124;&#xBA85;.

&#xC804;&#xAE30; &#xC774;&#xB3D9; &#xBCC4;&#xAC70; &#xADF8;&#xB9AC;&#xACE0; x-&#xC120; &#xD615;&#xAD11; &#xB9E4;&#xD551;&#xC5D0; &#xC758;&#xD574; metalloproteins&#xC758; &#xAC80;&#xCD9C;.

Todos los sistemas vivos dependen de metaloproteinas. Sin embargo, aunque herramientas para la separaci&oacute;n e identificaci&oacute;n de apo-prote&iacute;nas est&aacute;n bien desarrollados, los que permiten la identificaci&oacute;n y cuantificaci&oacute;n de metaloproteinas individuales dentro de mezclas complejas est&aacute;n a&uacute;n nacientes. Aqu&iacute;, describimos la separaci&oacute;n electrofor&eacute;tica de una mezcla de anhidrasa carb&oacute;nica, ceruloplasmina, ureasa y hemoglobina mediante electroforesis 2D nativa y radiograf&iacute;a fluorescencia asignaci&oacute;n-un enfoque hemos desarrollado ampliamente aplicables, no requieren equipos especializados para la preparaci&oacute;n de la muestra y parece ser ampliable en el futuro.

Separaci&oacute;n electrofor&eacute;tica y detecci&oacute;n de metaloproteinas por asignaci&oacute;n de fluorescencia de rayos x.

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the periplasmic metalloproteome was resolved in non-denaturing bidimensional gel electrophoresis, followed by X-ray fluorescence visualization and identification by mass-spectrometry. A single spot containing the electron shuttle protein azurin was responsible for the observed increments in cuproprotein contents. In agreement, lack of either Cu(+)-ATPase induced an increase in azu transcription. This is associated with an increase in the expression of anr and rpoS oxidative stress response regulators, rather than cueR, a copper sensing regulator. We propose that azurin overexpression and accumulation in the periplasm is part of the cellular response to cytoplasmic oxidative stress in P. aeruginosa.

Periplasmic response upon disruption of transmembrane Cu transport in Pseudomonas aeruginosa.

Metalloproteins are enormously important in biology. While a variety of techniques exist for studying metals in biology, X-ray absorption spectroscopy is particularly useful in that it can determine the local electronic and physical structure around the metal center, and is one of the few avenues for studying "spectroscopically silent" metal ions like Zn(II) and Cu(I) that have completely filled valence bands. While X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) are useful for studying metalloprotein structure, they suffer the limitation that the detected signal is an average of all the various metal centers in the sample, which limits its usefulness for studying metal centers in situ or in cell lysates. It would be desirable to be able to separate the various proteins in a mixture prior to performing X-ray absorption studies, so that the derived signal is from one species only. Here we describe a method for performing X-ray absorption spectroscopy on protein bands following electrophoretic separation and western blotting.

Lydia A. Finney

X-ray Science Division,
Advanced Photon Source,
X-ray Science Division, Advanced Photon Source

Argonne National Laboratory

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Lydia A. Finney

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}X-ray Science Division,Advanced Photon Source,X-ray Science Division, Advanced Photon Source

Argonne National Laboratory

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray Science Division,
Advanced Photon Source,
X-ray Science Division, Advanced Photon Source