Biochemistry, Biophysics, and Structural Biology Commons^™

Structure-Expression Relationships Of The 15-Kda Selenoprotein Gene, Easwari Kumaraswamy, Andrey Malyhk, Konstantin V. Korotkov, Sergei Kozyavkin, Yajun Hu, Byeong J. Lee, Dolph L. Hatfield, Alan M. Diamond, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stemloop structures resembling selenocysteine insertion …

Amino Acid Osmolytes In Regulatory Volume Decrease And Isovolumetric Regulation In Brain Cells: Contribution And Mechanisms, Herminia Pasantes-Morales, Rodrigo Franco, M. Eugenia Torres-Marquez, Karla Hernandez-Fonseca, Arturo Ortega

School of Veterinary and Biomedical Sciences: Faculty Publications

Brain adaptation to hyposmolarity is accomplished by loss of both electrolytes and organic osmolytes, including amino acids, polyalcohols and methylamines. In brain in vivo, the organic osmolytes account for about 35% of the total solute loss. This review focus on the role of amino acids in cell volume regulation, in conditions of sudden hyposmosis, when cells respond by active regulatory volume decrease (RVD) or after gradual exposure to hyposmotic solutions, a condition where cell volume remains unchanged, named isovolumetric regulation (IVR). The amino acid efflux pathway during RVD is passive and is similar in many respects to the volume-activated anion …

Method And Apparatus For Adaptive Filtering By Counting Acoustic Sample Zeroes In Ultrasound Imaging, Gregory R. Bashford, Edward D. Nonnweiler, David D. Becker, David John Muzilla

Biomedical Imaging and Biosignal Analysis Laboratory

An ultrasound imaging system having an adaptive spatial filter the filter coefficients of which, for particular image parameter sample, are determined by counting the number of neighboring image parameter samples having zero or near-zero values. If the number of zero or near-zero values in a data window is greater than a predetermined threshold, the data in the window is passed, not filtered. This filter has two advantages over other spatial filters. First, image parameter data samples having only zero or near-zero neighboring values (i.e., isolated "point noise") are not smeared. Second, boundaries such as the edge of color in a …

Structural And Functional Conservation At The Boundaries Of The Chicken Β-Globin Domain, Noriko Saitoh, Adam C. Bell, Felix Recillas-Targa, Adam G. West, Melanie A. Simpson, Michael Pikaart, Gary Felsenfeld

Department of Biochemistry: Faculty Publications

We show that the 3' boundary of the chicken β -globin locus bears striking structural similarities to the 5' boundary. In erythroid cells a clear transition in DNase I sensitivity of chromatin at the 3' end of the locus is observed, the location of this transition is marked by a constitutive DNase I hypersensitive site (HS), and DNA spanning this site has the enhancer-blocking capacity of an insulator. This HS contains a binding site for the transcription factor CTCF. As in the case of the 5' insulator, the CTCF site is both necessary and sufficient for the enhancer-blocking activity of …

Crystal Structure Of A Nonsymbiotic Plant Hemoglobin, Mark S. Hargrove, Eric Allen Brucker, Boguslaw Stec, Gautam Sarath, Raúl Arredondo-Peter, Robert V. Klucas, John S. Olson, George N. Phillips Jr.

Gautam Sarath Publications

Background: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O₂ conditions.
Results: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 Å resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal …

Method And Apparatus For Controlling Acoustic Signal Bandwidth In An Ultrasonic Diagnostic Imaging System, Jeffrey R. Resnick, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An ultrasonic imaging system includes a receive beamformer that generates analog receive signals and a scan converter. A receive signal processing path interconnects the receive beamformer and the scan converter, and this processing path included both an A/D converter characterized by a selectable sampling rate and at least one filter characterized by at least one filter parameter. The filter parameter is selected as a function of the sampling rate to provide enhanced image quality.

Insect Cellular Reactions To The Lipopolysaccharide Component Of The Bacterium Serratia Marcescens Are Mediated By Eicosanoids, Jon C. Bedick, R L. Pardy, R. W. Howard, David W. Stanley

Department of Biochemistry: Faculty Publications

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply …

Efflux Of Osmolyte Amino Acids During Isovolumic Regulation In Hippocampal Slices, Rodrigo Franco, Octavio Quesada, Herminia Pasantes-Morales

School of Veterinary and Biomedical Sciences: Faculty Publications

The efflux of potassium (K⁺) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (22.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited …

Method And Apparatus For Three-Dimensional Ultrasound Imaging Of Biopsy Needle, Syed Omar Ishrak, Mir Said Seyed-Bolorforosh, William Thomas Hatfield, Todd Michael Tillman, Brian Peter Geiser, Gregory R. Bashford, Michael Joseph Washburn

Biomedical Imaging and Biosignal Analysis Laboratory

A method and an apparatus for three-dimensional ultrasound imaging of a needle-like instrument, such as a biopsy needle, inserted in a human body. The instrument is visualized by transmitting ultrasound beams toward the instrument and then detecting the echo signals using a linear array of transducer elements. The problem of ultrasound being reflected from a biopsy needle in a direction away from the transducer array is solved by steering the transmitted ultrasound beams t increase the angle at which the beams impinge upon the biopsy needle. Ideally the ultrasound beams are perpendicular to the needle. This increases the system's sensitivity …

Isovolumic Regulation In Nervous Tissue: A Novel Mechanism Of Cell Volume Regulation, O. Quesada, R. Franco, K. Hernandez-Fonseca, K. Tuz

School of Veterinary and Biomedical Sciences: Faculty Publications

Cell volume regulation is a property present in most animal cell lineages that allows them to recover their original volume after events of swelling or shrinkage. Such events can be caused by changes in external osmolarity or to osmotic gradients generated during normal cell functioning.4,6 The mechanism of cell volume regulation involves transmembrane fluxes of osmotically active solutes in the necessary direction to counteract the net gain or loss of intracellular water.9 The process through which cells recover their normal volume after swelling is named Regulatory Volume Decrease (RVD). This consists of the efflux of inorganic osmolytes, such as K …

Rbcs Suppressor Mutations Improve The Thermal Stability And Co2/O2 Specificity Of Rbcl-Mutant Ribulose-1,5-Bisphosphate Carboxylaseyoxygenase, Yu-Chan Du, Seokjoo Hong, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

In the green alga Chlamydomonas reinhardtii, a Leu²⁹⁰-to-Phe (L²⁹⁰F) substitution in the large subunit of ribulose-1,5-bisphosphate carboxylaseyoxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala222-to-Thr and Val²⁶²-to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO₂/O₂ specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between β strands A and B of the Rubisco small subunit, which is …

Multiple Factors Independently Regulate Hila And Invasion Gene Expression In Salmonella Enterica Serovar Typhimurium, Robin L. Lucas, C. Phoebe Lostroh, Concetta C. Dirusso, Michael P. Spector, Barry L. Wanner, Catherine A. Lee

Department of Biochemistry: Faculty Publications

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more

about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In

addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the

pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence

of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the

PhoR-PhoB two-component regulatory system, suggesting that hilA expression may …

Multiple Factors Independently Regulate Hila And Invasion Gene Expression In Salmonella Enterica Serovar Typhimurium, Robin L. Lucas, C. Phoebe Lostroh, Concetta C. Dirusso, Michael P. Spector, Barry L. Wanner, Catherine A. Lee

Department of Biochemistry: Faculty Publications

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more

about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In

addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the

pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence

of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the

PhoR-PhoB two-component regulatory system, suggesting that hilA expression may …

Two-Step Processing Of Human Frataxin By Mitochondrial Processing Peptidase, Patrizia Cavadini, Jiri Adamec, Franco Taroni, Oleksandr Gakh, Grazia Isaya

Department of Biochemistry: Faculty Publications

We showed previously that maturation of the human

frataxin precursor (p-fxn) involves two cleavages by the

mitochondrial processing peptidase (MPP). This observation

was not confirmed by another group, however,

who reported only one cleavage. Here, we demonstrate

conclusively that MPP cleaves p-fxn in two sequential

steps, yielding a 18,826-Da intermediate (i-fxn) and a

17,255-Da mature (m-fxn) form, the latter corresponding

to endogenous frataxin in human tissues. The two cleavages

occur between residues 41–42 and 55–56, and both

match the MPP consensus sequence RX ↓ (X/S). Recombinant

rat and yeast MPP catalyze the pài step 4 and 40 …

Job Compensation In The Biotechnology Core Laboratory, Debra A. Mcmillen, William M. Old, Janice L. Bleibaum, Charles M. Nicolet, Katheryn R. Resing, Gautam Sarath, Satya P. Yadav

Gautam Sarath Publications

The results of a poll of core facility professionals by the Association of Biomolecular Resource Facilities (ABRF) Survey Committee.

Biochemical And Biophysical Analysis Of The Intracellular Lipid Binding Proteins Of Adipocytes, Melanie A. Simpson, Vince J. Licata, Natalie Ribarik Coe, David A. Bernlohr

Department of Biochemistry: Faculty Publications

Adipocytes express two lipid-binding proteins; the major one termed the adipocyte lipid-binding protein or aP2 (ALBP/aP2) and a minor one referred to as the keratinocyte lipid-binding protein (KLBP). In order to evaluate the potential physiological roles for these proteins, their biochemical and biophysical properties have been analyzed and compared. ALBP/aP2 and KLBP exhibit similar binding affinities for most long-chain fatty acids; however, ALBP/aP2 exhibits a two to three-fold increased affi nity for myristic, palmitic, oleic and linoleic acids, the predominant fatty acids of adipocytes. As measured by guanidinium hydrochloride denaturation, the stability of ALBP/aP2 is nearly 3 kcal/mol greater than …

Melanoma Chondroitin Sulphate Proteoglycan Regulates Cell Spreading Through Cdc42, Ack-1 And P130Cas, Kathryn M. Eisenmann, James B. Mccarthy, Melanie A. Simpson, Patricia J. Keely, Jun-Lin Guan, Kouichi Tachibana, Louis Lim, Ed Manser, Leo T. Furcht, Joji Iida

Department of Biochemistry: Faculty Publications

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumors. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α4β1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine- phosphorylated p130cas, an adaptor protein important in tumor cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of …

New Mammalian Selenocysteine-Containing Proteins Identified With An Algorithm That Searches For Selenocysteine Insertion Sequence Elements, Gregory V. Kryukov, Valentin M. Kryukov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Mammalian selenium-containing proteins identified thus far contain selenium in the form of a selenocysteine residue encoded by UGA. These proteins lack common amino acid sequence motifs, but 3’-untranslated regions of selenoprotein genes contain a common stem-loop structure, selenocysteine insertion sequence (SECIS) element, that is necessary for decoding UGA as selenocysteine rather than a stop signal. We describe here a computer program, SECISearch, that identifies mammalian selenoprotein genes by recognizing SECIS elements on the basis of their primary and secondary structures and free energy requirements. When SECISearch was applied to search human dbEST, two new mammalian selenoproteins, designated SelT and SelR, …

Method And Apparatus For Controlling Acoustic Signal Bandwidth In An Ultrasonic Diagnostic Imaging System, Jeffrey R. Resnick, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An ultrasonic imaging system includes a receive beam former that generates analog receive signals and a scan converter. A receive signal processing path interconnects the receive beamformer and the scan converter, and this processing path includes both an A/D converter characterized by a selectable sampling rate and at least one filter characterized by at least one filter parameter. The filter parameter is selected as a function of the sampling rate to provide enhanced image quality.

An Insulator Element And Condensed Chromatin Region Separate The Chicken Β-Globin Locus From An Independently Regulated Erythroid-Specific Folate Receptor Gene, Marie-Noelle Prioleau, Pascale Nony, Melanie A. Simpson, Gary Felsenfeld

Department of Biochemistry: Faculty Publications

We have identified a folate receptor gene upstream of the chicken β-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of β-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the β-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient …

Redox Regulation Of Cell Signaling By Selenocysteine In Mammalian Thioredoxin Reductases, Qi-An Sun, Yalin Wu, Francesca Zappacosta, Kuan-Teh Jeang, Byeong Jae Lee, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The …

Posttranscriptional Regulation Of Mammalian Methionine Synthase By B12, Sumedha Gulati, Lawrence C. Brody, Ruma V. Banerjee

Department of Biochemistry: Faculty Publications

Methionine synthase is one of two key enzymes involved in the removal of the metabolite, homocysteine. Elevated homocysteine levels constitute a risk factor for cardiovascular diseases and for neural tube defects. In cell culture, the activity of methionine synthase is enhanced several-fold by supplementation with its cofactor, B₁₂. The mechanism of this regulation is unknown, although it has been ascribed to a shift from apoenzyme to holoenzyme. Using sensitive assay techniques as well as a combination of Northern and Western analyses, we demonstrate that the effect of B,, on induction of methionine synthase activity is paralleled by an …

Optimal Replication Activity Of Vesicular Stomatitis Virus Rna Polymerase Requires Phosphorylation Of A Residue(S) At Carboxy-Terminal Domain Ii Of Its Accessory Subunit, Phosphoprotein P, Leroy N. Hwang, Nathan Englund, Tapas Das, Amiya K. Florida, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites …

Nucleotide Sequences For Detection Of Serpulina Hyodysenteriae, Gerald E. Duhamel, Robert Elder

School of Veterinary and Biomedical Sciences: Faculty Publications

The invention provides a method for detecting the presence of Serpulina hyodysenteriae in a biological Sample, an oligonucleotide primer and an S. hyodysenteriae-specific oligonucleotide probe useful in that method, and an article of manufacture that contains the primers and/or probe. Also provided are an about 2.3-kb DNA fragment derived from genomic DNA of S. hyodysenteriae and encoding for an about 56 kDa polypeptide, a recombinant expression vector containing the DNA fragment, the 56 kDa polypeptide and a monoclonal antibody reactive with the peptide, and a method of assaying for antibodies reactive with the 56 kDa peptide.

Levels Of Major Selenoproteins In T Cells Decrease During Hiv Infection And Low Molecular Mass Selenium Compounds Increase, Vadim Gladyshev, Thressa C. Stadtman, Dolph L. Hatfield, Kuan-Teh Jeang

Vadim Gladyshev Publications

It has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using ⁷⁵Se-labeled human Jurkat T cells, we show that the levels of four ⁷⁵Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells. These major selenoproteins migrated as 57-, 26-, 21-, and 15-kDa species on SDS/PAGE gels. In our earlier studies, the 57-kDa protein was purified from T cells and identified as a subunit of thioredoxin reductase. …

Yeast And Human Frataxin Are Processed To Mature Form In Two Sequential Steps By The Mitochondrial Processing Peptidase, Steven S. Branda, Patrizia Cavadini, Jiri Adamec, Frantisek Kalousek, Franco Taroni, Grazia Isaya

Department of Biochemistry: Faculty Publications

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich’s ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not …

Plasmd Bearing A Cdna Copy Of The Genome Of Bovine Viral Diarrhea Virus, Chimeric Derivatives Thereof, And Method Of Producing An Infectious Bovine Wral Darrheavirus Using Sad Plasmid, Ruben O. Donis, Ventzislav B. Vassilev

School of Veterinary and Biomedical Sciences: Faculty Publications

A plasmid bearing a cDNA copy of the genome of bovine viral diarrhea virus (BVDV), chimeric derivatives of the plasmid and a method of producing an infectious bovine viral diarrhea virus using the plasmid are disclosed. The invention relates to a plasmid DNA molecule that replicates easily in E. coli and contains a sufficient portion of the genome of BVDV, cloned as cDNA, to be a suitable template to produce RNA in vitro which, upon transfection into bovine cells, gives rise to infectious BVDV. The BVDV created by the process of the invention can be engineered for use as a …

In Situ Hybridization For The Detection And Localization Of Swine Chlamydia Trachomatis, C. Chae, D.-S. Cheon, D. Kwon, O. Kim, B. Kim, J. Suh, D. G. Rogers, K. D. E. Everett, A. A. Anderson

School of Veterinary and Biomedical Sciences: Faculty Publications

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4–7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were …

Early And Late Endosomal Compartments Of Entamoeba Histolytica Are Enriched In Cysteine Proteases, Acid Phosphatase And Several Ras-Related Rab Gtpases, Lesly A. Temesvari, Ed Harris, Samuel L. Stanley Jr., James Cardelli

Department of Biochemistry: Faculty Publications

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding …

Affinity Chromatography: A Review Of Clinical Applications, David S. Hage

David Hage Publications

Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLCbased methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with …