CST Guide:

24
Section III: Workflow Tools
Screening & Quantification
using ELISA to screen and quantify
cellular responses at the target level
How can I quantify levels of total and post-translationally
modified proteins in response to cell stimuli
The sandwich enzyme-linked immunosorbent assay (ELISA) is a quantitative, microplate-based assay
that detects changes in abundance of a target protein within a cell sample using a capture antibody
coated to the plate and a detection antibody to generate signal. Unlike a western blot that achieves
specificity by using a single antibody combined with separation of protein by size, an ELISA is highly
specific because it uses two antibodies recognizing different epitopes of the same protein. ELISAs are
broadly utilized in both research and clinical settings; here, we focus on their application in research for
the study of intracellular signaling using phospho-specific and total protein antibodies. ELISAs can also
be used to quantify epigenetic changes using histone acetylation- or methylation-specific antibodies.
The quantitative nature, high sensitivity, plate-based format, and ease-of-use make ELISAs a commonly
used tool for screening in drug discovery and development.
Sandwich ELISAs can be used to:
• Screen therapeutic compounds for effects on post-translational modification of target proteins
• Measure relative changes in total or post-translationally modified protein levels in response
to an external stimuli such as a therapeutic compound
• Assess effects of a cell stimulus at multiple time points or dosages
• Quantitatively measure activation of signaling proteins in a high throughput format
• Confirm data generated by western blot or other antibody-based applications
For example, the PathScan ® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit from Cell Signaling Technology
(CST) was used to assess phosphorylation of Bcl-2 at Ser70 in Jurkat cells treated with a single dose
of paclitaxel. As shown in the figure, paclitaxel treatment increases phosphorylation of Bcl-2 at Ser70
more than 2-fold relative to untreated control without affecting the level of total Bcl-2 protein. The
changes quantified by ELISA were independently observed by western blot.
Assessing the effects of growth factor or cytokine stimulation using ELISA
PathScan ® Phospho-Akt (Thr308) Chemiluminescent
Sandwich ELISA Kit #7135: Relationship between protein
concentration of lysates from untreated and PDGF-treated
NIH/3T3 cells and immediate light generation with chemiluminescent
substrate is shown. Cells (80% confluence)
were treated with PDGF #9909 (50 ng/ml) and lysed after
incubation at 37ºC for 5 min. Graph inset corresponding to
the shaded area shows high sensitivity and a linear response
at the low protein concentration range.
PDGF-treated
Untreated
RLU (x10 5 )
60
50
40
30
20
10
0
0.0
PathScan ® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit #7300: Treatment
of HeLa cells with IFN-α stimulates phosphorylation of Stat3 at Tyr705, detected
by #7300, but does not affect the level of total Stat3 protein, detected by a Stat3
antibody via western analysis. OD 450 readings are shown in the top figure, while
the corresponding western blot using Phospho-Stat3 (Tyr705) (3E2) Monoclonal
Antibody #9138 or Stat3 antibody, is shown in the figure to the right.
Untreated
IFN-α-treated
70
0.1
0.2 0.3 0.4 0.5 0.6
Protein conc. of lysate (mg/ml)
Absorbance 450nm
4
3
2
1
0
14
10
6
2
Phospho-Stat3
(Tyr705)
chapter 24: screening & Quantification
0.02 0.06 0.1
0.7 0.8
Phospho-
Stat3
(Tyr705)
Stat3
– + IFN-α
How PathScan ® Sandwich ELISA Kits can benefit your research:
• Highly Sensitive: detect low abundance target proteins and generate measurable linear
responses at low lysate concentrations
• Rigorously Validated Antibody Pairs: generate accurate data and minimize background,
ensuring reliable results
• Multiple Detection Options: colorimetric or chemiluminescent detection options are available
• Specialized Packaging: bulk orders and custom 384-plate size are available
Paclitaxel treatment of Jurkat cells results
in phosphorylation of Bcl-2 at Ser70.
High sensitivity ELISAs produce a broad dose response
and are linear in low concentration ranges.
PathScan ® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit #11874:
Treatment of Jurkat cells with Paclitaxel stimulates phosphorylation of Bcl-2
at Ser70, as detected by #11874, but does not affect the levels of total Bcl-2
detected by PathScan ® Total Bcl-2 Sandwich ELISA Kit #12030. Jurkat cells
were untreated or treated with λ phosphatase or Paclitaxel #9807 (1 mM,
20 hr, 37°C). The absorbance readings at 450 nm are shown in the top
figure, while the corresponding western blots using Phospho-Bcl-2 (Ser70)
(5H2) Rabbit mAb #2827 (left panel) or Bcl-2 (D55G8) Rabbit mAb (Human
Specific) #4223 (right panel) are shown in the bottom figure.
λ phosphatase-treated
Untreated
Paclitaxel-treated
Absorbance 450nm
4
3
2
1
0
Phospho-Bcl-2
(Ser70)
Total Bcl-2
PathScan ® Phospho-EGF Receptor (Tyr1068) Sandwich ELISA
Kit #7240: The relationship between protein concentration of lysates
from untreated and EGF-treated A-431 cells and kit assay optical
density readings. After starvation, A-431 cells (85% confluence) were
treated with EGF #8916 (100 ng/ml, 5 min, 37°C) and then lysed.
EGF-treated
Control
2.5
1.5
1.0
0.5
Absorbance 450nm 2.0
0
0.05 0.10 0.15 0.20 0.25
Protein conc. of lysate (mg/ml)
0.30
– – +
+ – –
– – +
+ – –
Paclitaxel
λ Phosphatase
In a second example, the PathScan ® Phospho-Akt (Thr308) Chemiluminescent Sandwich ELISA Kit
#7135 was used to examine the effects growth factor stimulation on phosphorylation of Akt at Thr308,
an indicator of Akt activation and signaling. As shown in the figure, PDGF-mediated activation of Akt
was quantified along a range of lysate levels, demonstrating the kit’s high sensitivity and a linear
response at low protein concentrations. A similar assessment of the effects of cytokine stimulation on
phosphorylation of Stat3 at Tyr705 was performed using #7300.
272 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
CST Technical Support
When you contact CST for technical support, you can be confident you will be working with a colleague you can trust,
striving to provide the highest quality products and services to you, our customer. Please see our Technical Support
resource page online for contact information. www.cellsignal.com/cstsupport
www.cellsignal.com/screening
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