SPI-Mark™ Colloidal Gold Reagents
Choice of Gold Conjugate
Gold conjugates may be used directly or indirectly to label antigens in cells
and tissues. In the direct method the gold is conjugated to the primary
antibody, whether monoclonal or polyclonal. This allows a single incubation to
be performed and provides the simplest detection system. In the indirect
method a primary unlabelled antibody is applied to the specimen to locate the
antigen. This is followed by a gold labelled secondary antibody that detects the
primary antibody. This gold labelled secondary antibody is almost always an
affinity purified polyclonal. In this way an amplification of the signal is achieved,
often up to 10 times compared with a direct incubation, according to the
particle size. If, for example, the primary antibody is from mouse (a
monoclonal) then the second gold labelled antibody will be Goat anti-Mouse. A
full range of BBI secondary gold labelled antibodies is given, together with full
guidelines for making the correct choice, in Choosing a Gold Conjugate. The
indirect method for immunolabeling is the most common for studying antigens
in cells and tissues and avoids the need to label every primary antibody. It
provides a universal method for detecting any primary antibody from the same
primary species, i.e. all rabbit antibodies may be detected by Goat anti-Rabbit
gold conjugates, etc. The indirect method is longer than the direct method
because of the need for two or three separate incubations but is the more
widely used. The protocols described below use the indirect method exclusively.
The choice of gold particle size depends upon the magnification which is to be
used in the EM. In principle smaller gold particles produce a higher labeling
intensity due to their greater concentration in solution and the lower steric
hindrance produced. For example an IgG may be approximately 8nm in length.
Gold particles of 1nm or 5nm produce very little steric hindrance to the
antibody activity, while larger particle sizes reduce the potential labeling
intensity due to their sheer size. Larger particle sizes, however, are more easily
seen at lower magnifications. For those users undecided about which particle
size to choose for preliminary investigations, 10nm gold conjugates are
preferred since these may be clearly seen over a wide range of magnifications in the EM.
For ultra high sensitivity the very smallest, 1nm gold conjugates may be
preferred since the number of gold particles attaching to the tissue will be very
high. This requires that extensive washing following the incubations is
necessary. These particles are invisible, however, at magnifications below
500,000x and will need to be silver enhanced. Silver enhancing allows the
particles to grow to any size suitable, the reaction stopped simply by washing
in water. Silver enhancing procedures for EM applications are described below
and are identical to those for LM use.
For LM studies, where silver enhancing is required, the choice is between 1nm
and 5nm gold conjugates. 1nm conjugates provide very intense labeling and
can penetrate cell membranes during pre-embedding routines. However very
thorough washing is required to prevent unwanted background following the incubations.
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Thursday May 17, 2012
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