SPI-Mark™ Colloidal Gold Reagents
Instructions for Use and Sample Protocols
Incubation Buffer (Buffer A)
A typical incubating buffer suitable for both EM and LM incubations, which
provides the components for eliminating the sources of background may be as follows:
PBS or TBS
+1% Normal serum (e.g. goat serum if using Goat anti-rabbit IgG: gold)
+0.1% Tween 20
+1% BSA
+0.1% sodium azide (NaN3)
Adjusted to pH8.2.
This is referred to as buffer A in the following text and is typical of the Buffer
required for each step of an incubation.
The PBS or TBS provides the tonicity for antibody reaction. Antibodies will not
work in the absence of adequate salt.
The normal serum covers tissue sites that may otherwise attract the gold
labelled antibodies non-specifically.
The BSA blocks other "sticky" tissue components, which would otherwise
attract the antibodies non-specifically.
The Tween 20 reduces the possibility of hydrophobic attraction between tissue
components and the gold particles.
The pH of 8.2 is often preferred to reduce background caused by charge
attraction of gold particles to positively charged tissue components. At higher
pH values there is less positive charge on proteins in the tissue and thus less
attraction for negatively charged gold particles. This is usually the main reason
for non-specific background and is cured simply by raising the pH of the
incubating buffer.
In order to achieve the highest possible signal with the lowest possible nonspecific
background it is important to be aware of all the factors in both
specimen preparation and in subsequent incubations which can affect these
results. For the indirect, two step method of immunolabeling attention must be
given to the quality of the antibodies, the necessary dilution of each one, the
buffers used, and the incubation protocol. Wherever possible the method
should be kept as simple as possible and each step or component must be
questioned as to its value. There is no point adding many ingredients to a
buffer in order to avoid background if no such background exists. It may
actually cause the introduction of unwanted background!
To Ask a Question or Make a Comment
To Place an Order or Request a Quote
Return to:
Thursday May 17, 2012
© Copyright 2006 - 2012. By Structure Probe, Inc.
Contacting SPI Supplies and Structure Probe, Inc.
All rights reserved.
All trademarks and trade names are the property of their respective owners.
Privacy Policy
Worldwide Distributors, Representatives, and Agents
