rev: May 14, 2008
Return Home page
BACK (Master GOLD PAGE)
Return (Gold Enhancing Reagents)
Silver Enhancing Kit-Biochemical(*) CAT#
RDI-SEKB250, new cat#: 55R-SEKB250
Price: $412.50
The Silver Enhancing Kit is a simple to use and sensitive method for
enhancement of immunogold labelled antigens on a solid support. It depends on
the reduction of silver from solution in the presence of a developer by the
gold label attached to proteins/antibodies/nucleic acids mounted on a solid
phase for direct viewing by eye.
Although the intensification kit is relatively insensitive to light during the
time of incubation, it should be stored at 4C and in the dark or frozen over
the long term. When properly stored the reagents are stable for at least 6
months at 4C or in-efinitely at -20C.
STORE AT 4C OR FROZEN AND AWAY FROM EXCESSIVE HEAT AND LIGHT.
The kit has been specially formulated to combine ease of use with good sensitivity and contrast. The components of the solutions have been selected to allow good control over time of development with minimal background. Because of its relative insensitivity to light the enhancement may be monitored in normal room lighting during development. The specimens need no fixing after development but may simply be thoroughly washed in tap water. The development operates at pH 7.4 to minimise the difficulties experienced with other enhancing methods where very low pH sometimes causes the extraction of labelled proteins from the specimen.
If contact with the solutions is made by the skin simply wash the area under tap water.
PROCEDURE
1. The specimens should be labelled with the appropriate immuno gold reagents for biochemical application. Both 1nm and 20nm gold conjugates are recommended for application to antigens immobilised on a solid phase support. 20nm gold conjugates offer the advantage of greater visibility of the gold label before enhancement with silver. Greater intensity of labeling may be achieved with the 1nm gold conjugates due to the larger number of gold particles attaching to the antigen. The technical instruction booklet describing gold labelling procedures and provided with these products.
2. After labelling with gold conjugates the solid phase support should be
thoroughly washed in deionised or distilled water. In all cases non metallic
labware should be employed. Avoid the use of metallic holders and forceps since
metals will cause silver precipitation out of solution. A plastic tray is ideal
for incubations.
3. Mix equal quantities of initiator and enhancer together, enough to cover the surface of the solid phase support to a depth of 3-4mm when in the pastic tray. The development may be monitored in normal room lighting conditions. Avoid performing the reaction to strong lighting or in direct sunlight.
4. The development of a silver deposit at the site of gold labelling is
dependent on temperature. At 20 DEG C development time is approximately 15-20
minutes for the enhancement of 20nm gold conjugates. With 1nm gold conjugates a
typical enhancing time is 30-40 minutes. Lower temperatures take longer, higer
temperature require a shorter time. The solutions have been formulated tro
allow the user to easily control the time of development. In may cases the
incubation can be left for an hour or more. The development should be stopped
when specific staining is complete and background is still minimal. The
reaction is stopped by washing immediatley in tap water for 2-3 minutes. No
fixing is required.
A color change will be seen as the reduction of silver from solution occurs on
the solid phase support. The specifically labelled sites will change from invisible-yellow-orange-light
brown-dark brown-black.
If required the procedure may be continued by simply using freshly mixed solutions.
After thorough washing in water allow the support to dry in air.
TEST STRIPS (*)
Provided with this kit is a pack of test strips that have been dot blotted with gold conjugate serially diluted to provide a decreasing protein content. There are 5 spots carrying gold particles conjugated to 10ng, 1ng, 100pg, 10pg and 1pg of protein. The sensitivity of the kit may be tested simply by adding 5-10 drops of both initiator and enhancer together in the tube. The spots will change from pink or invisible-yellow-orange-light brown-dark brown-black. All 5 spots will eventually be stained (in about 20-30 minutes). The strips are intended to be used to judge the stability of the kit after long term storage. Further packs of test strips are availabe. Pack of 10 strips -CAT # SETS10
Freezing:
A great advantage of this kit is that it may be stored at 4 DEG C or frozen.
Simply place the kit in a depp freeze for almost indefinite storage. The kits
have been thoroughly tested for at least 10 freeze thzw cycles and work
perfectly. At 4 DEG C stability is guaranteed for at least 6 months if properly
stored in the dark with caps securely screwed on.
For In Vitro Research Use Only
RDI Division of Fitzgerald Industries Intl
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com