The typical way of labeling antibodies for immunohistochemical detection is by indirect conjugation. A two-step process is used – a primary antibody is raised against the antigen, and then a second, fluorescently-labeled antibody is used to detect the first.
Evidently, a one-step staining method would be far simpler and quicker than the indirect method. However, until recently direct conjugation has been avoided because of problems in sensitivity and amplification of the signal. Factors influencing this include the sample size needed, low percentage of recovery and dilution.
The indirect method has greater sensitivity, as the signal can be amplified through using several secondary antibodies, each specific to a different antigenic site on the primary protein. However, the procedure is tedious, requiring numerous incubation and wash steps. Using secondary proteins also means more likelihood of cross-over problems and non-specific binding.
We at Novus Biologicals supply Lightning-Link direct labeling kits to scientists. Lightning-Link has overcome earlier barriers to direct labeling, while offering benefits over indirect methods.
The technique involves covalently binding the primary antibody with alkaline phosphatase (AP), which is held in a proprietary binding solution. In combination with the antibody to be labeled, proprietary reagents in the Lighting-Link solution are activated to achieve gentle and directional coupling with AP at a near-neutral PH.
Lightning-link avoids the desalting and dialysis steps of indirect protocols. There is no excessive conjugate dilution, and small quantities of protein can be recovered to 100%. Adding Lightning-Link to our antibody catalogue enables full conjugation of primary antibodies to be achieved within 30 seconds.
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