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FIGURE 1.4
Monomeric quantal brightness determination for receptor tagged with fluorescent protein.
FIGURE 1.5
Monomeric quantal brightness determination for antibody complex labeling using a reference
monomeric fluorescent protein.
In our experiments, mGPF is often used for assessing monomeric QB. A trans-
fected construct is expressed and detected with a primary antibody from the same
species as the protein of interest. For example, if the primary antibody targeting
the receptor of interest is raised in the mouse, then a control cell expression sample
can be made with farnesylated mGFP, which will then also be labeled using a mouse
antibody. The same fluorescently tagged secondary antibody will then be used to
visualize the primary antibodies in both samples. Just remember that to apply this
method, one has to be selective in the choice of the fluorophore conjugated to the
secondary antibodies to ensure clear spectral separation from that of the monomeric
fluorescent protein tag ( Fig. 1.5 ).
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