CST Guide:

13
Section II: ANTIBODY APPLICATIONS
chapter
Sandwich ELISA
The enzyme-linked immunosorbent assay (ELISA) is a method for detecting a specific analyte in a
heterogeneous biological fluid or cell extract. ELISA is a quantitative immunoassay typically perfomed
in multi-well microplates, which allows for simultaneous analysis of multiple samples. In a sandwich
ELISA, the target molecule is bound between a capture antibody immobilized on a microplate and a
detection antibody that is detected by a secondary antibody conjugated to an enzymatic or other reporter
system. The use of two antibodies recognizing different epitopes of the same protein results in a highly
specific assay. Sandwich ELISA assays are useful for the screening of small molecule inhibitors without
using expensive instrumentation or complex data analysis methods. Sandwich ELISA output can be
measured by a number of optical methods, including colorimetric (absorbance) and chemiluminescent
(RLU or relative light units). Chemiluminescent readouts show a higher dynamic range, especially at low
target protein concentrations. The specifcity and sensitivity of the capture and detection antibodies are
critical to ensuring high signal-to-background ratios. The capture and detection antibody pairs are also
amenable to customization on plate-based and bead-based platforms.
Rapid cell harvest and elevated phosphatase
inhibitor concentrations may improve ELISA
results for low abundance proteins.
While it is important to achieve complete cell lysis for the investigation of both total and post-translationally
modified protein targets, rigorous extraction methods must be balanced with preservation of
the antibody-antigen interactions required for ELISA. Preserving the phosphorylation state of target
epitopes can be more challenging when working with specific cell lines or tissues, as some cells
produce higher levels of phosphatases than others. When investigating the phosphorylation state of
low abundance proteins or targets that are difficult to detect, especially in high phosphatase-producing
cell lines, it is sometimes necessary to replace the standard protocol for preparation of cell lysates
with a rapid harvest method. This method omits the cell scraping and sonication steps and uses a lysis
buffer that contains increased concentrations of Ser/Thr phosphatase inhibitors. For example, when
initial ELISA results from phospho-targets produce a low signal, scientists at CST use a no scrape, no
sonicate rapid harvest method combined with a lysis buffer (#7018) that contains increased amount of
phosphatase inhibitors*, as a means to increase target-specific signal.
13: Sandwich eLISA
*Complete protocol and buffer components
can be found on the PathScan ®
Sandwich ELISA Lysis Buffer #7018
product datasheet
Improved ELISA detection
of phospho-AMPKα (Thr172)
was achieved using an
alternative lysis buffer and
a rapid extraction protocol.
3
2.5
2
1.5
1
0.5
PathScan ® Phospho-AMPKα
(Thr172) Sandwich ELISA Kit #7959:
C2C12 cells, untreated or treated with
H 2 0 2 (10 mM for 10 min), were lysed and
harvested using Cell Lysis Buffer (10X)
#9803 or PathScan ® Sandwich ELISA
Lysis Buffer #7018 (which contains
inhibitors of Ser/Thr phosphatases) and
detected using #7959. Equal protein
concentrations of cell extract were used
in each assay. ELISA signal was greatest
when cell lysis was performed with
#7018 and a rapid harvest procedure
with no cell scraping or sonication.
0
Scrape/Sonicate No Scrape/Sonicate Scrape/Sonicate No Scrape/Sonicate
#9803
#7018
Cell Lysate total protein concentration (0.25 mg/ml)
Untreated
H 2 O 2 Treated
Phosphatase
inhibitors preserve
the constitutive phosphorylation
of Mer.
ELISA Tips for Success
For detection of intracellular targets, the success of an ELISA experiment depends critically on the
quality and composition of the cell lysis buffer and the method used to generate the cell extract.
Include protease and/or phosphatase inhibitors in lysis
buffer to preserve protein levels and phosphorylation state.
Using fresh cell lysis buffer containing appropriate protease and phosphatase inhibitors ensures that
changes in the protein or phosphorylation levels are not due to proteolytic degradation of target proteins
or loss of phosphorylation due to endogenous phosphatases.
Absorbance 450nm
3
2
1
0
Total Mer
Phosho-Mer (panTyr)
– + – +
phosphatase
inhibitor
PathScan ® Phospho-Mer (panTyr) Sandwich ELISA Kit #13340: Constitutive
phosphorylation of Mer in HCC827 cells lysed in the presence of protease inhibitors
and phosphatase inhibitors (phospho lysate) is detected by #13340 (upper, right).
In contrast, a low level of phospho-Mer protein is detected in HCC827 cells lysed
in the presence of protease inhibitors and absence of phosphatase inhibitors
(nonphospho lysate). Similar levels of total Mer protein from both nonphospho and
phospho lysates are detected by PathScan ® Total Mer Sandwich ELISA Kit #13488
(upper, left). Absorbance at 450 nm is shown in the top figure while corresponding
western blots using Mer (D21F11) XP ® Rabbit mAb #4319 (left) and a phospho-Mer
(Tyr749/753) antibody (right) are shown in the bottom figure. Phosphatase inhibitors
include sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate.
Nonphospho
Phospho
PathScan ® Sandwich ELISA Colorimetric Protocol
NOTE: Refer to product-specific datasheet or product webpage for assay incubation temperature.
A. Solutions and Reagents
For lyophilized formulation
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1. Microwell strips: Bring all to room temperature before use.
2. Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green
solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully
reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of
Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.
3. HRP-Linked Antibody: Supplied lyophilized as a red colored cake or powder. Add 1.0 ml of HRP Diluent (red solution) to
yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute.
To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP
Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C. NOTE: Some PathScan ®
ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
4. Detection Antibody Diluent: Green colored diluent for reconstitution and dilution of the detection antibody (11 ml provided).
5. HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‐Linked Antibody (11 ml provided).
6. Sample Diluent: Blue colored diluent provided for dilution of cell lysates.
7. 1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan ® Sandwich ELISA Kit) in purified water.
8. 1X Cell Lysis Buffer: Prepare 1X lysis buffer using either PathScan ® Sandwich ELISA buffer (ready to use 1X stock, #7018)
or 10X Cell Lysis Buffer (#9803). To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml 10X Cell Lysis Buffer to 9 ml of dH 2 O,
mix. Both buffers can be stored at 4°C for short-term use (1–2 weeks). Recommended: Add 1 mM phenylmethylsulfonyl
fluoride (PMSF) (#8553) immediately before use. NOTE: Refer to product-specific datasheet or webpage for lysis buffer
recommendation.
9. TMB Substrate: (#7004)
10. STOP Solution: (#7002)
220 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstelisa
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