Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels.

Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel.

Western blotting is one of the few basic techniques widely used in the study of proteins in life science research. Despite its prevalence, the procedure has remained practically unchanged for more than 20 years. Although the method is viewed as being error-prone and as requiring excessive hands-on time, it is still widely accepted because it provides sensitive and direct information about the protein characteristics.

Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer.

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming.

Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel.

Western blotting is one of the few basic techniques widely used in the study of proteins in life science research. Despite its prevalence, the procedure has remained practically unchanged for more than 20 years. Although the method is viewed as being error-prone and as requiring excessive hands-on time, it is still widely accepted because it provides sensitive and direct information about the protein characteristics.

Photophysical properties of a new DyLight 594 dye.

We describe spectral properties of novel fluorescence probe DyLight 594. Absorption and fluorescence spectra of this dye are in the region of Alexa 594 fluor spectra. The quantum yield of DyLight 594 in conjugated form to IgG is higher than corresponding quantum yield of Alexa 594 by about 50%.

Solid-phase biotinylation of antibodies.

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent.

Coated microwell plate-based affinity purification of antigens.

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes.