CST Guide:

Section III: Workflow Tools
Small GTPase Detection Assays
Like second messenger assays, small GTPase detection assays allow a researcher to evaluate cell
signaling by measuring the presence of activated Ras GTPase as a readout for signaling activity. The
Ras GTPase superfamily consists of five protein families (Ras, Rho, Rab, Arf, and Ran) whose members
cycle between the active, GTP-bound, and inactive, GDP-bound, state. GTPases play an important role
in cell cycle progression, cell survival, and actin cytoskeletal organization and are actively studied in
many fields, including tumor biology.
• GTPase Detection Assays measure activation of a single GTPase in the cell using a GST-fusion
protein to bind the activated form of GTP-bound Rac1 (or other GTPase), which can then be
immunoprecipitated with glutathione resin and analyzed by western blot.
GTPase detection assays assess levels
of active GTPase in a cell lysate.
Active Rac1 Detection Kit #8815: NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro
with GTPγS or GDP to activate or inactivate Rac1 (refer to optional step C in protocol). The lysates
were then incubated with glutathione resin and GST-PAK1-PBD (lanes 2 and 3). GTPγS-treated
lysate was also incubated without GST-PAK1-PBD in the presence of glutathione resin as a
negative control (lane 4). WB analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples
(lanes 2–4) was performed using a Rac1 Mouse mAb. Anti-mouse IgG, HRP-linked Antibody
#7076 was used as the secondary antibody.
1 2 3 4
– + – – GDP
– – + + GTPγS
– + + – GST-PAK1-PBD
26
Verification
using siRNA-mediated knockdown to verify function
How can I ask a highly targeted question about protein
or antibody function in the context of the whole cell
Small interfering RNAs (siRNA) are short segments (19–27 nucleotides) of double stranded RNA that
silence expression of a single gene in a sequence-specific manner. This is achieved when a single
strand of the RNA duplex associates with the RNA-induced silencing complex (RISC), a multiprotein
complex containing endonucleases that bind and degrade mRNA at a sequence complementary to the
siRNA. Thus, transfection of a specific siRNA molecule into living cells can result in specific inhibition of
expression of the corresponding protein.
siRNA can be used to:
• Verify an antibody is detecting its intended target
• Analyze the functional role of a node within a signaling pathway
• Validate a potential therapeutic target
• Explore gene function in the context of a disease or biological system
The GTP-bound
GTPase pull-down
process can be
divided into 3 steps.
Step 1: Mix sample, binding protein,
and glutathione resin in the spin cup
and incubate at 4ºC to allow GTP-bound
GTPase binding to the glutathione resin
through GST-linked binding protein.
Step 2: Remove unbound proteins by
centrifugation.
Step 3: Elute glutathione resin-bound
GTPase with SDS buffer. The eluted
sample can then be analyzed by WB.
sample
spin cup
collection tube
= Other Proteins
= GTP-bound GTPase
= GDP-bound GTPase
= Binding Protein
= Glutathione
Step 1 Step 2
Step 3
- mix sample with resin
and binding protein
- incubate
wash
elute
Western blot
For example, in the experiment below, SignalSilence ® p27 Kip1 siRNA from Cell Signaling Technology is
used to verify antibody specificity. The robust signal obtained from p27 Kip1 (D69C12) XP ® Rabbit mAb
#3686 in the presence of control siRNA (-) is diminished when p27 Kip1 expression is silenced using
two different p27 Kip1 siRNAs (+), indicating the antibody specifically detects the p27 Kip1 protein.
siRNA-mediated knockdown
verifies p27 Kip1
antibody specificity.
kDa
80
60
50
40
30
20
50
40
I
II
– + +
p27
Kip1
β-Actin
p27 Kip1
siRNA
SignalSilence ® p27 Kip1 siRNA I #12324
and siRNA II #12410: WB analysis of extracts
from HeLa cells, transfected with 100 nM
SignalSilence ® Control siRNA (Unconjugated)
#6568 (-), #12324 (+), or #12410 (+), using p27
Kip1 (D69C12) XP ® Rabbit mAb #3686 (upper)
or β-Actin (D6A8) Rabbit mAb #8457 (lower). The
p27 Kip1 (D69C12) XP ® Rabbit mAb confirms
silencing of p27 Kip1 expression, while the β-Actin
(D6A8) Rabbit mAb is used as a loading control.
SignalSilence ® siRNA
Experimental Controls
SignalSilence ® Control siRNA
(Fluorescein Conjugate) #6201
Transfection efficiency
SignalSilence ® Control siRNA
(Unconjugated) #6568
siRNA specificity
How SignalSilence ® siRNA can benefit your research:
• Two siRNAs Available for Most Targets: confirm inhibition at the functional protein
level using two equally potent sequences
• Rigorously Validated Duplexes: confidently inhibit expression of your intended protein
• Appropriate Controls: monitor transfection efficiency with a fluorescein-labeled
nontargeted siRNA and specificity with an unconjugated control
• Target Proteins From Different Species: inhibit expression of human or mouse targets
A positive control
siRNA confirms c-Myc
siRNA specificity in the
target cells, suggesting
that absence of c-Myc
expression is due to
specific knockdown.
SignalSilence ® Control
siRNA (Unconjugated) #6568:
Confocal IF analysis of HeLa
cells, transfected with #6568
(left) or SignalSilence ® c-Myc
siRNA I #6341 (right), using
c-Myc (D84C12) XP ® Rabbit mAb
#5605 (green). Actin filaments
were labeled with DyLight 554
Phalloidin #13054 (red).
276 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/verification 277