Technical Considerations for Contemporary Western Blot Techniques.

Western blotting continues to be a workhorse assay in laboratories throughout the world. The utility, low cost and accessibility of western blotting have allowed the technique to remain in practice, despite being developed over 40 years ago. Advances in antibody specificity, chemiluminescent formulations, properties of fluorescent molecules and imaging techniques provide gains in sensitivity, dynamic range, and ease of use.

Direct kinetic fingerprinting and digital counting of single protein molecules.

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte.

Multiple analyte profiling (MAP) index as a powerful diagnostic and therapeutic monitoring tool.

A robust data mining algorithm is presented as a critical solution to the challenge of managing intensive data generated from the recently developed multiplexing techniques, which allow simultaneous detection of up to 500 biomarkers in a few microliters of a single sample. Furthermore, detailed methodology is provided for exploiting the new algorithm along with examples for description of the first application as a powerful diagnostic and therapeutic monitoring tool in the management of breast cancer, as a disease model.

Method to quantify cytokines and chemokines in mouse brain tissue using Bio-Plex multiplex immunoassays.

This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample.

Multiplexing techniques for measurement of the immunomodulatory effects of particulate materials: Precautions when testing micro- and nano-particles.

Particulate materials at nano- and micro-scales have widespread pharmaceutical and medical applications. Understanding the interactions of these materials with biological systems is crucial for the design of clinically-viable biomaterials of high safety profiles. Immunomodulatory effects of particulate materials can be studied via multiplexing techniques that are capable of measuring up to 500 biomarkers in a few microliters of biological samples.

V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting.

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process.

Beyond molecular beacons: optical sensors based on the binding-induced folding of proteins and polypeptides.

Many polypeptides and small proteins can be readily engineered such that they only fold upon binding a specific target ligand. This approach couples target recognition with a considerable change in polymer structure and dynamics. Recent years have seen the development of a number of biosensors that couple these large changes to readily measurable optical (fluorescent) outputs.

Chimeric peptide beacons: a direct polypeptide analog of DNA molecular beacons.

We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.

Peptide beacons: a new design for polypeptide-based optical biosensors.

Both epitope mapping and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here, we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to a macromolecular target.

Excimer-based peptide beacons: a convenient experimental approach for monitoring polypeptide-protein and polypeptide-oligonucleotide interactions.

While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets.