Biomedical subjects

Daryl Emery

Publications and source records attributed to Daryl Emery.

3 recordsLinked to original sources

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

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

Use of Immunomatrix Methods to Improve Protein-Protein Interaction Detection.

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 immunoprecipitation and coimmunoprecipitation is that the conditions used to elute the precipitated antigen also release the antibody, 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. 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 yields IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chains contamination.

Journal Article↗