APPLICATION NOTE

REPROBE YOUR IMMUNOASSAY

SAMPLES USING REPLEX WITH JESS

INTRODUCTION

The identification and quantification of protein expression levels is critical to understanding biological
processes like those that drive cancer and other diseases. Unfortunately, limited samples sizes are far
too common in these research areas, leaving researchers desiring more data from their limited samples. To
measure protein expression by traditional methods like the Western blot, researchers must sacrifice most, if not all,
of their sample for only a few data points, which are poorly reproducible and semi-quantitative at best. To overcome these limitations,
Simple Western™ with Jess™ is an automated, capillary-based immunoassay that provides highly reproducible and quantitative data
with a tiny amount of material. With just 3 µL of sample, Jess offers pg-level sensitivity on up to 25 samples in 3 hours, all in a fully
automated fashion. Now, Jess comes with RePlex™ so that you can reuse the same 3 µL of sample for automated sequential rounds
of probing.

REPLEX WITH SIMPLE WESTERN LETS YOU DO MORE WITH LESS

To gain the most data out of your precious sample, Jess provides a two-step immunoassay that is
performed within the same capillary. This new feature, called RePlex removes the antibodies from the
first round of probing to perform either a second round of probing with new antibodies or total protein
detection. Importantly, RePlex efficiently removes antibodies between probing without compromising
the integrity of the immobilized protein or its epitopes, allowing for excellent reproducibility across
cycles. With Jess’s chemiluminescence and fluorescence channels, you can detect multiple targets per
cycle. The second cycle can also be dedicated to total protein detection so that you can normalize your
data with confidence. All the steps of RePlex are automatically performed with Jess, providing more data,
and lowering the cost of reagents and consumables per result.

PUTTING REPLEX TO THE TEST

In this Application Note, we show how you can use RePlex to detect and quantify pan and phospho isoforms of proteins such as
those in the PI3K/Akt signaling pathway. This pathway modulates cell growth, survival, and apoptosis, and it is frequently altered in
human cancers, contributing resistance to radiation and chemotherapy treatment1,2. First, we provide evidence that RePlex efficiently
removes antibodies between probing without compromising the integrity of the immobilized protein or its epitopes. Then we use
RePlex to multiplex antibodies to phosphorylated isoforms and pan isoforms of Akt in the first cycle, followed by a second cycle to
normalize the data by total protein detection. Finally, we extend RePlex to characterize concomitant downstream targets and protein
expression in different tissue types.
MATERIALS AND METHODS PROTEIN DETECTION IS UNAFFECTED
BETWEEN REPLEX CYCLES
Experiments were performed with the products listed in Table 1
and antibodies listed in Table 2. RePlex™ with Jess™ enables two-step immunoassays that rely on
the removal of antibodies from the first round of probing (Probe
TABLE 1 1) prior to a second round of probing with fresh antibodies (Probe
PRODUCT VENDOR
PRODUCT 2). To demonstrate that signal intensity and reproducibility are not
NUMBER
compromised between Probe 1 and Probe 2, we measured the
12-230 kDa Jess/Wes Separation
ProteinSimple SM-W004 signal intensity of AKT1 and AKT2 antibodies in MCF7 lysate in
Module
the two probing steps. Lane view showed similar band intensities
Anti-Rabbit Detection Module ProteinSimple DM-001 (Figure 1A), while quantitation of AKT1 and AKT2 confirmed
Anti-Mouse Detection Module ProteinSimple DM-002 that peak areas were very similar for Probe 1 and Probe 2 when
Anti-Mouse NIR Detection Module ProteinSimple DM-009 looking at each protein target (Figure 1B). Regardless of the
order in which each protein was probed, AKT1 and AKT2 in MCF7
Total Protein Detection Module ProteinSimple DM-TP01
lysates detected in Probe 1 or Probe 2 in a RePlex assay showed
RePlex Kit ProteinSimple RP-001 excellent reproducibility and similar signal intensity across both
Jurkat Cell Extract
Cell Signaling
9273 probing cycles. This gives us confidence that protein integrity
Technologies
and re-probing are not compromised by antibody removal in
MCF7 Cell Extract
Cell Signaling
34499 RePlex assays.
Technologies
Lung Human Whole Tissue Lysate Novus Biologicals NB820-59239
Colon Human Whole Tissue Lysate Novus Biologicals NB820-59205 A Probe 1 Probe 2
Kidney Human Whole Tissue Lysate Novus Biologicals NB820-59231
Cap 2 3 4 14 15 16 5 6 7 17 18 19
Brain Human Whole Tissue Lysate Novus Biologicals NB820-59177 kDa
180-
Liver Human Whole Tissue Lysate Novus Biologicals NB820-59232
116-
Breast Human Whole Tissue Lysate Novus Biologicals NB820-59203
TABLE 1. Products used in this study. 66-
AKT2

40-

TABLE 2
PRODUCT DILUTION
ANTIBODY VENDOR
NUMBER FACTOR
Cell Signaling 1:100 (Chemi) 12-
Mouse Pan AKT 58295
Technologies 1:50 (NIR)
Cell Signaling 180-
Rabbit Phospho-AKT(Ser473) 9271 1:50
Technologies
116-
Cell Signaling
Rabbit Phospho (Thr308) 9275 1:50
Technologies 66-
AKT1
Cell Signaling
Rabbit Phospho-AKT1(Ser473) 9018 1:50
Technologies
40-
Cell Signaling
Rabbit Phospho-AKT2(Ser473) 8599 1:50
Technologies
Cell Signaling
Rabbit AKT1 2938 1:100
Technologies
12-
Cell Signaling
Rabbit AKT2 3063 1:100
Technologies

Mouse AKT3 Antibody

Cell Signaling
8018 1:50
B AKT1 and AKT2 peak area in Probe 1 and Probe 2
Technologies

TABLE 2. Antibodies used in this study. Dilution factors used in the study are listed. 1400000
Probe 1
Average peak area

1200000 Probe 2
Sample, reagent, antibody, and assay plate preparation were 1000000
performed as described in the Simple Western product inserts.
800000
Lysates were diluted 1:10 in Sample Buffer prior to mixing with
600000
fluorescent standards. Target proteins were immunoprobed
400000
with a primary antibody followed by HRP-labeled secondary
antibody for amplified chemiluminescent detection, or NIR- 200000
labeled secondary antibody for fluorescent detection. The 0
AKT1 AKT2
antibodies from the first immunoassay were removed using the
RePlex reagent, followed by a second immunoassay or Total FIGURE 1. Signal intensity and reproducibility for AKT1 and AKT2 in MCF7 lysates,
detected in Probe 1 or Probe 2 in a RePlex assay. (A) Lane view in Compass for
Protein Assay in the same capillary. For the Total Protein Assay, Simple Western shows similar band intensities, and (B) quantitation shows
immobilized proteins were biotinylated and later detected with comparable peak areas for AKT1 and AKT2 in each probing cycle. Error bars
Streptavidin-HRP. represent standard deviations of the means.
2
ANTIBODIES ARE EFFICIENTLY REMOVED
WHEN USING REPLEX™ WITH JESS™

A critical step in RePlex is the efficient removal of antibodies after 2 versus the original signal in Probe 1. From this analysis, we
the first round of probing. Thus, we assessed removal efficiency found that greater than 98% of signal was removed for pan AKT
of antibodies against pan and phosphorylated AKT on Jurkat and (Figure 2A). The lane view and removal efficiency for multiple
MCF7 lysates. To do so, the primary and secondary antibodies AKT antibodies are shown in Figures 2B and 2C, respectively. All
from Probe 1 were removed in the automated assay. The same antibodies tested showed greater than 96% removal efficiency,
secondary antibody that was used in Probe 1 was then used in with most of the antibodies tested showing 98% or higher. These
Probe 2 without the addition of new primary antibody to detect data provide evidence that a second round of antibody probing
any residual primary antibody from Probe 1. The removal efficiency may be performed with little to no crossover from the first round
was measured as the percent of signal that was detected in Probe of probing.

A 70,000

Chemiluminescence P1,2
600,000 60,000
50,000
40,000
500,000 Pan AKT 30,000
20,000
Chemiluminescence P1,2

10,000
400,000
0
MW (kDa) 66

300,000

200,000 Residual Pan AKT

100,000

40 66 116
MW (kDa)

B Jurkat
C
MCF7
pSerAKT AKT1 AKT2 pSerAKT1 pGSKb panAKT AKT1 pGSK3b Probe 1 removal efficiency
Probe: 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
kDa
180-
110%

100%
116-
90%
66-
80%

70%

40- 60%

50%
Phospho Ser AKT

AKT1

AKT2

Phospho AKT2

Phospho AKT1

Phospho GSK3b

AKT1

AKT2

Pan AKT

Phospho GSK3b

12-

Jurkat MCF7

FIGURE 2. First antibody probes are efficiently removed during the RePlex assay. (A) More than 98% of the signal from Probe 1 was removed for mouse pan AKT antibody.
(B) Lane view shows the immunoassay signal for Probe 1 and no residual signal for Probe 2 for multiple targets in Jurkat and MCF7 cell lines. (C) Removal efficiencies (%) for
these targets were calculated as (Peak area Probe 1 – Peak area Probe 2)/Peak area Probe 1 x 100. Error bars represent standard deviations of the means.

3
IMMUNOASSAY AND TOTAL PROTEIN
DETECTION SEAMLESSLY BACK-TO-BACK

RePlex™ with Jess™ performs sequential immunoassays AKT proteins detected using chemiluminescence, as indicated by
seamlessly back-to-back. In a similar manner, it can also be used black bands, and pan AKT using NIR detection, as indicated by
to perform an immunoassay in the first round and total protein the red bands in Probe 1. Total protein detection from Probe 2 is
detection in the same capillary. To demonstrate this, we used shown in blue. Figure 3B shows representative electropherograms
MCF7 cells untreated and treated with human IGF1 (hIGF1). We of the signal for the various AKT antibodies and total protein
were able to detect pan and phosphorylated AKT in Probe 1 by signal. Automated normalization of phosphorylated and pan
multiplexing the primary and secondary antibodies and then AKT signal to total protein signal is displayed in the Peaks Table
subsequently detect total protein signal in Probe 2 after removal of in Compass for Simple Western (Table 3).
the antibodies. The lane view in Figure 3A shows phosphorylated

A B 3,000
Probe 1 NIR: Pan AKT 5,000 Phospho AKT Pan AKT
MCF7 + hIGF1 (Ser 473)
2,000
1,000
Ser Thr
Probe 1 Chemi: 0 0
473 308
3,000
5,000 Phospho AKT Pan AKT
MCF7 Control

Fluorescence NIR P1
Chemiluminescence P1

hIGF1: + – + – (Ser 473)

2,000
kDa 1,000
0 0

3,000
Phospho AKT Pan AKT
MCF7 + hIGF1
5,000
2,000
Probe 1: (Thr 308) 1,000

Pan AKT 0 0

Phospho AKT Phospho AKT Pan AKT

3,000

MCF7 Control
5,000
2,000
(Thr 308)
1,000
0 0
12 40 66 116 180 230
MW (kDa)

6,000
Total Protein
MCF7 + hIGF1
4,000

2,000

0
6,000

Probe 2: MCF7 Control

Chemiluminescence P2

4,000

Total Protein 2,000

0
6,000

MCF7 + hIGF1
4,000

2,000

0
6,000

MCF7 Control
4,000

2,000

0
12 40 66 116 180 230
MW (kDa)

FIGURE 3. Automated immunoassay and total protein detection in a single capillary of AKT phosphorylation in MCF7 lysates treated and untreated with hIGF1. (A)
Phosphorylated AKT (phospho AKT) isoforms and pan AKT were detected in Probe 1 using chemiluminescence and NIR fluorescence, respectively, while total protein signal
was detected in Probe 2. (B) Example graph views of phospho AKT and pan AKT (top), and total protein signal (bottom) for samples in panel A.

TABLE 3. Peaks Table in Compass for Simple Western shows automated normalization of phosphorylated and pan AKT signal to total protein signal demonstrates
quantitation of target protein expression.
4
MULTI-TARGET DETECTON USING REPLEX™
In addition to probing the same target with different antibodies, high primary antibody removal efficiency of RePlex, the same
we also used RePlex to characterize other signaling proteins secondary antibody species could be used in both Probe 1 and
along with AKT detection, including cRaf and GSK3 beta Probe 2, without any carryover signal from Probe 1. This is seen
(GSK3b). Using lysates from control and LY294002-treated Jurkat in the electropherograms in Panel B where only a single peak is
cells, we could clearly detect changes in phosphorylation status detected for phosphorylated GSK3b in Probe 2, the blue line,
for multiple proteins in the same sample, as shown in Panel A and no residual peak is present from the phosphorylated AKT
and quantified in Panel C in Figure 4. Furthermore, due to the antibody that was used in Probe 1 even though both primary
antibodies are rabbit.

A B 100,000
16,000
pG KT

SK T

pc AKT

pc KT
pS 3b

3b
pG rAK
A

A
SK

pS f

af
90,000
a
er

er

er
e

R
pS

pS

14,000
80,000
Chemiluminescence P1 Probe 2 Probe 1

Chemiluminescence P2
12,000
Phospho Phospho 70,000
10,000 GSK3b Ser AKT 60,000

8,000 50,000

40,000
6,000
30,000
4,000
20,000
2,000 10,000

0 0

MW (kDa)
66

C Fold decrease in phosphorylation of

GSK3b and cRaf
5
4
Fold dercease

3
2
Probe: 1 2 1 2 1 2 1 2 1
Treatment: Ctl Ly Ctl Ly 0
pGSK3b pcRaf

FIGURE 4. RePlex detection of concomitant phosphorylation for AKT and downstream targets in the same capillary. (A) Lane view of AKT, GSK3b and cRaf phosphorylation
in control (Ctl) and LY294002-treated (LY) Jurkat lysates. (B) RePlex enables use of the same secondary antibody in Probe 1 and Probe 2, with no carryover signal, as
demonstrated by detection of phosphorylated AKT and GSK3b using rabbit-derived primary antibodies in sequential probes of the same sample. (C) Quantitation shows a
decrease in GSK3b and cRaf phosphorylation in LY294002-treated Jurkat lysates. Error bars represent standard deviations of the means.

5
As final demonstration of the utility of RePlex™, we characterized and Probe 2 for each sample (Figure 5A), while the graph shows
phosphorylation status of AKT in multiple tissue types. Figure 5 phosphorylation signal normalized to pan AKT signal (Figure 5B).
includes detection of pan and serine or threonine phosphorylated As might be expected, different tissues showed both different
AKT in the same capillary for multiple tissue types, including liver, levels of AKT expression and phosphorylation.
kidney, colon, brain, breast, and lung. Lane view shows Probe 1

A Tissue: Liver Kidney Colon Brain Breast Lung B AKT phosphorylation level in various tissue
Phospho Site: S T S T S T S T S T S T normalized to the level of AKT expression
kDa
0.8
0.7
0.6

phospho/pan ratio
0.5
Probe 1
0.4
Phospho AKT
0.3
0.2
0.1
0

Phospho Ser AKT

Phospho Thr AKT

Phospho Ser AKT

Phospho Thr AKT

Phospho Ser AKT

Phospho Thr AKT

Phospho Ser AKT

Phospho Thr AKT

Phospho Ser AKT

Phospho Thr AKT

Phospho Ser AKT

Phospho Thr AKT

Brain Breast Colon Kidney Liver Lung
Probe 2
Pan AKT

FIGURE 5. RePlex assays detecting phospho-Ser473 AKT and pan AKT or phospho-Thr308 AKT and pan AKT in the same capillary show variation in phosphorylation status
for multiple tissue types and reduces the amount of tissue sample required for multi-target detection. (A) Lane view and (B) corresponding quantitation of phosphorylation
for each tissue sample. Phosphorylation signal was normalized to pan AKT signal from the same capillary. Error bars represent standard deviations of the means. (S, Ser473;
T, Thr308)

6
REPLEX IS THE FUTURE OF SEQUENTIAL IMMUNOASSAYS

The strip-and-reprobe technique in traditional Western blot is widely used to gain more information from a single blot, thus saving
on precious or costly samples. However, this technique is well known to suffer from unpredictable protein loss and typically requires
lengthy optimization to achieve optimal stripping efficiency and maximize protein retention on the blot. This makes any quantitative
approach using standard strip and reprobe techniques nearly impossible.

By contrast, the RePlex™ assay developed for the Simple Western™ platform Jess™ is an optimized and automated two-step
immunoassay that enables even more data to be generated per sample. Optimization of the RePlex approach has shown that
signal intensities and reproducibility for the same protein before and after antibody removal were not affected. Therefore, antibody
removal using RePlex does not cause protein loss and does not affect epitope binding, which is crucial for quantitation in sequential
immunoassays. We attribute this to the proteins being covalently immobilized to the capillary surface in Simple Western assays, unlike
protein association with membranes used in western blots, which is driven via hydrophobic interactions.

We have demonstrated here highly effective antibody removal and excellent target protein retention in the sample capillary for
multiple targets in the AKT/PI3K pathways using RePlex. Phosphorylation, isoform and pan specific antibodies were used for a subset
of targets to assess the overall degree of phosphorylation and evaluate the isoform specific expression levels and phosphorylation
states in RePlex assays. Both cell lines as well as tissue lysates were analyzed in chemiluminescence and NIR fluorescence modes
using RePlex on Simple Western to evaluate multiple targets in the same sample, including phosphorylation level for specific targets
relative to either target protein expression or total protein content. This is especially important when differently treated samples must
be analyzed independently and compared. Lastly, the highly effective antibody removal in RePlex, even for targets with high signal,
allows detection of multiple proteins in one sample using the same antibody species without carryover signal, significantly expanding
the antibody pairings used in Probe 1 and Probe 2.

In summary, the development of the RePlex assay for Simple Western combines the time-saving benefits of an automated immunoassay
with multi-target detection and protein normalization in the same capillary, resulting in more data and quantitation with less sample.

REFERENCES

1. Akt regulates cell survival and apoptosis at a postmitochondrial

level, H Zhou, XM Li, J Meinkoth and RN Pittman, Journal of Cell
Biology, 2000; 151(3):483-94.

2. The protein kinase PKB/Akt regulates cell survival and apoptosis

by inhibiting Bax conformational change, H Yamaguchi and HG
Wang, Oncogene, 2001; 20:7779-86.

Jess
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