Vector Laboratories offers a number of reagents for the
detection of blotted protein samples. These reagents can
recognize epitopes, fusion protein tags, or carbohydrate
groups in the targeted protein. The choice of reagents will
depend on the nature of the target protein and the chosen
visualization method. Sensitivity, reproducibility, and ease of
use are important considerations when choosing detection
One-Step Fluorescence Detection. Fluorochrome conjugated
secondary antibodies may be used to detect an unconjugated
primary antibody on a blot. These fluorescent detection
reagents are used in conjunction with the proper membrane
and visualization equipment. This method is fast and simple
and allows multiple targets to be visualized simultaneously.
Fluorescent reagents are listed and described in detail in “Immunofluorescence”. This section focuses on the
more common enzyme-based detection methods.
One-Step Enzymatic Detection. One-step enzymatic detection
offers greater sensitivity and more versatility due to the types
of detection reagents and enzyme substrates available. For
one-step detection, alkaline phosphatase (AP) and peroxidase
(HRP) conjugated secondary antibodies are available.
Amplified Detection with Biotin/Avidin and Biotin/Streptavidin
Systems. Biotin/avidin or biotin/streptavidin detection systems
offer the greatest sensitivity by signal amplification and the
greatest flexibility in detection methods. The VECTASTAIN®
ABC-AmP™ kits, for example, are complete detection systems
that offer sensitivity, reliability, and versatility. Streptavidin or
avidin based reagents can detect a variety of macromolecules
on the blot: biotinylated secondary antibodies,
biotinylated antibodies against fusion protein tags,
biotinylated lectins, and even biotinylated target
proteins blotted directly onto the membrane.
Enzyme Substrates. Substrates that produce either a
visible chromogenic precipitate or a chemiluminescent/
chemifluorescent signal can be used for visualization.
Alkaline phosphatase allows sustained substrate conversion
and increased sensitivity with low background. Peroxidase
has faster reaction kinetics resulting in a more rapid signal
development. Vector Laboratories offers a variety of
proprietary peroxidase and alkaline phosphatase substrates
that not only satisfies sensitivity requirements and personal
preferences, but also allows multiple targets to be visualized
simultaneously on the blot. In addition, our DuoLuX™
Chemiluminescent/Chemifluorescent substrates provide highly
sensitive chemiluminescent and chemifluorescent visualization.