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Monitoring Transfection Efficiency
Because low transfection efficiency is the most frequent
cause of unsuccessful gene silencing experiments, optimizing transfection
conditions is critical. The Silencer Gene Specific siRNA Controls
are ideal for this application, particularly when they are fluorescently
labeled using the simple procedure provided with the Silencer siRNA
Labeling Kits. Once transfected into cells, uptake of the labeled siRNA
can be correlated to gene silencing by fluorescence microscopy using one
of Ambion's new primary antibodies matched to our gene specific siRNA
controls (Figure 1).
Figure 1. Characteristics of Ambion's Primary Antibodies for siRNA Research.
Primary Antibodies Matched to Silencer
Gene Specific Controls
Ambion provides mouse monoclonal antibodies for the detection of GAPDH,
c-myc, and -actin by immunofluorescence. Each of these primary antibodies
has been used in immunofluorescence experiments at Ambion to detect the
reduction in protein levels induced by siRNA. An example of such an experiment
is shown in Figure 2. (Antibodies are supplied in solution in a 100 g
unit size and are validated for use in immunofluorescence experiments.
Note that the concentration and volume will vary by antibody).
Figure 2. Following the Silencing of -actin. An siRNA targeting
-actin was labeled with Cy3 using the Silencer siRNA Labeling
Kit. The labeled siRNA was transfected into HeLa cells and cells were
analyzed 96 hours later. Green: -actin protein detected with anti--actin
(Ambion) and a fluorescein labeled secondary antibody. Red: Cy3 labeled
siRNA. Blue: DAPI stained nuclei. (Cy3 is a trademark of Amersham Biosciences.)
Ordering Information
Cat#
Product Name
Size
1632
Silencer siRNA Labeling Kit - Cy3
65 g labeled siRNA
1634
Silencer siRNA Labeling Kit - FAM
65 g labeled siRNA
4300
anti-GAPDH, mouse monoclonal
6C5
100 g
4302
anti-beta-actin, mouse monoclonal AC-15
100 g
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