Biomedical subjects

M Walid Qoronfleh

Publications and source records attributed to M Walid Qoronfleh.

7 recordsLinked to original sources

PubMed2006

Role and challenges of proteomics in pharma and biotech: technical, scientific and commercial perspective.

Contemporary proteomics, currently in its exponential growth phase, is a bewildering array of tools. Proteomic methods are the result of a convergence of rapidly improving mass spectrometry technologies, protein chemistry and separation sciences, genomics and bioinformatics. Strides in improving proteomics technologies to map and measure proteomes and subproteomes are being made. However, no single proteomic platform appears ideally suited to address all research needs or accomplish ambitious goals satisfactorily. However, proteomics is in a unique position to contribute to protein discovery and to public health in terms of better biomarkers, diagnostics and treatment of disease. While the potential is great, many challenges and issues remain to be solved. Fundamental issues, such as biological variability, pre-analytic factors and analytical reproducibility, remain to be resolved. Neither an all-genetic approach nor an all-proteomic approach will solve biological complexity. Proteomics will be the foundation for constructing and extracting useful knowledge to pharma and biotech depicted in the following path: data --> structured data --> information --> information architecture --> knowledge --> useful knowledge.

Animals↗

PubMed2005

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA.

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.

3T3 Cells↗

PubMed2004

Development of a high-throughput plate-based chemiluminescent transcription factor assay.

Transcription factors are DNA-binding proteins that regulate the expression of specific genes by controlling transcription initiation. Two families of transcription factors, NFkappaB and AP-1, play pivotal roles in controlling important cellular processes ranging from normal cell growth and differentiation to apoptosis and cancer. Identifying changes in the DNA-binding activity of these factors is essential to understanding the regulation of these processes. We have developed a high-throughput DNA-based ELISA capable of monitoring activated levels of NFkappaB (p50 and p65) and AP-1 (c-Jun and c-Fos). This chemiluminescent assay utilizes a 96-well plate format, eliminating the throughput challenges imposed by traditional gel shift assays and exceeding the sensitivity and dynamic range of standard colorimetric detection systems. The sensitivity of this assay enables distinction between subtle as well as dramatic differences in the DNA-binding activity of these factors that result from the treatment of cells with various inhibitors or activating agents.

Animals↗

PubMed2003

Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin.

We have developed an affinity-precipitation technique to facilitate conducting glutathione S-transferase (GST) pull-down assays. The dehydrated immobilized glutathione resin format, when combined with microcentrifuge spin columns, is a powerful tool that enables the simultaneous performance of resin hydration, the binding of the GST fusion protein, and the pull-down step with the appropriate protein partner in a semihigh-throughput fashion (multiple samples processed at the same time). The entire assay process is shortened and recovery is enhanced when coupled with a spin-column format, providing a convenient way to study protein-protein interactions. We successfully tested the resin format/technique in three common pull-down applications utilizing radiolabeled, overexpressed, and activated endogenous interacting protein partners.

Biochemistry↗

PubMed2003

Improved immunomatrix methods to detect protein:protein interactions.

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.

Animals↗

PubMed2003

SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry.

Albumins↗

PubMed2002

Development of a chemiluminescence-based ribonuclease protection assay.

The ribonuclease protection assay (RPA) is a widely used method for the detection and quantification of specific mRNA transcripts in a complex mixture of total RNA or mRNA molecules. While exhibiting many advantages over other RNA detection methods, RPAs are traditionally performed using radiolabeled probes that often require gel purification steps and lengthy exposure times to visualize results. Moreover, these probes can only be used for 1-2 weeks because of their short isotopic half-life and radiolysis. We report a method that improves the traditional RPA by replacing radiolabeled probes with biotinylated probes and lengthy exposure times with quick, streptavidin/HRP-based chemiluminescent detection technology. Biotinylated probes can be used without get purification and are stable for years, as opposed to weeks. Most importantly, our streptavidin/HRP-based chemiluminescent technology enables us to achieve sensitivity results similar to radioactive RPAs and to detect multiple transcripts in a single sample more efficiently. Furthermore, this new protocol addresses and eliminates the one major drawback unique to using biotinylated probes in chemiluminescent RPAs: a confounding artifact, not seen when running radioactive RPAs but commonly detected when using certain biotinylated rare message probes.

Actins↗