General protein-protein cross-linking.

This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization).

Western blotting using chemiluminescent substrates.

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane.

Protein biotinylation.

Biotin is a naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because biotin is small (244 Da), it can be conjugated to many proteins without altering their biological activities. The biotinylated molecule can be detected in ELISA, dot blot, or Western blot methods (see Western Blotting using Chemiluminescent Substrates) using streptavidin or avidin probes.

Performing and optimizing Western blots with an emphasis on chemiluminescent detection.

Immunodetection refers to any detection method that exploits the interaction of an antibody and antigen. The choice of detection method, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, depends on the researcher's preferences and requirements. If a researcher wants to quantify a low-abundance target protein then a chemiluminescent ELISA is used. If a researcher wants to identify a protein that is in high abundance, a colorimetric Western blot will suffice. If there are multiple targets within an assay, then multiplex fluorescence is typically used. This article focuses on Western blotting. Although colorimetric and fluorescent detection methods are discussed, chemiluminescent detection is used most often and is, therefore, discussed in great detail. Included is specific information about the chemiluminescent signal and factors that affect its intensity and longevity. We also describe types of blotting and present data and suggestions for obtaining semiquantitative data. Although classical Western blotting is typically used for qualitative purposes, we present information about effective quantitative analysis using specific controls. Common occurrences within the methodology and their possible explanations are also detailed. One frequent result is the appearance of ghost bands, which, based on our research, can be caused by high amounts of target or antibody cross-reactivity. Also included are the basic Western blot protocol and protocols for troubleshooting common problems and optimizing many of the specific factors that influence results.