SPI-Mark™ Colloidal Gold Reagents
Immunoblotting of Nucleic Acids and Proteins (BL Grade)
Blotting applications using BBI Gold Conjugates include the demonstration of
antigens, antibodies, proteins, macromolecules, total DNA/RNA, and
DNA/RNA fragments and gene sequences transferred to an immobilising
matrix. The procedures for transfer of these proteins and nucleotide sequences
follow the well established methods of Western and Southern blotting, or direct
dot blotting.
For Southern or Northern blots, for example, individual fragments of DNA or
RNA, as produced by restriction enzyme digestion, are separated by gel
electrophoresis and then transferred to an inert support such as nylon or
nitrocellulose. The immobilised DNA/RNA fragments can then be analysed by
in-situ hybridisation using a suitable biotinylated cDNA probe.
For Western blotting, protein dispersions from one or two dimensional gels, or
from dot blots of proteins, are immobilised onto a nitrocellulose sheet and the
whole sheet, or part of it, is incubated in the appropriate primary antibody.
In all cases, BBI Gold Conjugates of 1nm or 20nm particle diameter are used
to visualise the immobilised materials following incubation with an appropriate
primary antibody or probe. The 1nm gold conjugates give much higher
labeling intensity but must be silver enhanced to be visible. The 20nm gold
particles are directly visible on the membrane. The 1nm conjugate also
requires extensive washing to remove unbound particles from the membrane.
Because of the inexpensive and highly concentrated nature of the BL Grade
conjugates, large volumes may be used at high dilutions. Southern blots from
DNA gels are best immobilised on nylon based membranes where binding is
more efficient Proteins bind more effectively onto nitrocellulose. With nylon
membranes, however, it is necessary to block unoccupied sites more
extensively after blotting to avoid background staining.
A simple protocol for staining blotted protein or DNA is given below. Blotted
proteins, antibodies or antigens may be examined by the simple two step
indirect labeling method. DNA/RNA fragments, labelled with biotinylated
probes may be detected by using a gold labelled antibody to biotin.
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Thursday May 17, 2012
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